Targeted mutagenesis of the murine transferrin receptor-2 gene produces hemochromatosis

Abstract

Hereditary hemochromatosis (HH) is a common genetic disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. The vast majority of individuals with HH are homozygous for mutations in the HFE gene. Recently a second transferrin receptor (TFR2) was discovered, and a previously uncharacterized type of hemochromatosis (HH type 3) was identified in humans carrying mutations in the TFR2 gene. To characterize the role for TFR2 in iron homeostasis, we generated mice in which a premature stop codon (Y245X) was introduced by targeted mutagenesis in the murine Tfr2 coding sequence. This mutation is orthologous to the Y250X mutation identified in some patients with HH type 3. The homozygous Tfr2 Y245X mutant mice showed profound abnormalities in parameters of iron homeostasis. Even on a standard diet, hepatic iron concentration was several-fold higher in the homozygous Tfr2 Y245X mutant mice than in wild-type littermates by 4 weeks of age. The iron deposition in the mutant mice was predominantly hepatocellular and periportal. The mean splenic iron concentration in the homozygous Tfr2 Y245X mutant mice was significantly less than that observed in the wild-type mice. The homozygous Tfr2 Y245X mutant mice also demonstrated elevated transferrin saturations. There were no significant differences in parameters of erythrocyte production including hemoglobin levels, hematocrits, erythrocyte indices, and reticulocyte counts. Heterozygous Tfr2 Y245X mice did not differ in any measured parameter from wild-type mice. This study confirms the important role for TFR2 in iron homeostasis and provides a tool for investigating the excess iron absorption and abnormal iron distribution in iron-overload disorders.iron ͉ liver ͉ gene targeting T ransferrin receptor (TFR)2 is a recently identified type II membrane protein with significant homology to the classical transferrin receptor (TFR1). Both TFR1 and TFR2 are capable of transporting transferrin-bound iron into the cell (1) and supporting cell growth (2). However, their properties differ in several critical ways. TFR2 has a lower affinity for holotransferrin than does TFR1 (1, 3). The expression pattern of TFR2 mRNA is quite distinct from TFR1 as well (4, 5). In particular, the TFR2 gene is expressed at much higher levels in liver compared with other tissues, whereas the TFR1 gene demonstrates little hepatic expression. TFR1 and TFR2 differ also in their response to changes in cellular iron status. The TFR1 transcript contains multiple iron-responsive elements in the 3Ј-untranslated region. These elements stabilize the TFR1 transcript under conditions of low cellular iron. The TFR2 transcript does not contain these elements, and TFR2 message and TFR2 protein levels vary little with changes in iron status (4, 5). The observation that hepatic expression of Tfr2 persists despite iron overload in a murine Hfe gene knockout model of hereditary hemochromatosis (HH) led us to suggest that Tfr2-mediated iron uptake contributes...

Hematopoietic deletion of transferrin receptor 2 in mice leads to a block in erythroid differentiation during iron‐deficient anemia

“…1A, no visible differences in iron deposition in the livers of the KO and control mice was observed. These results indicated appropriate systemic iron regulation in mice even with a heterozygous deletion of Tfr2, as described previously [11,12].…”

“…Previous studies have shown that haplo-insufficiency of TFR2 does not alter systemic iron metabolism and a single allele in the heterozygous animals is sufficient to maintain appropriate iron levels [11,12]. It has been suggested that the Vav-Cre allele could be expressed in the ovaries, hence increasing the chance of germ-line transmission through the females [25,26].…”

“…Transferrin receptor 2 (TFR2) is involved in the regulation of iron metabolism; patients and mice harboring TFR2 mutations develop type III hereditary hemochromatosis, characterized by inappropriate hepcidin expression relative to body iron levels [9][10][11][12]. Analysis of mice with a hepatocyte-specific deletion of Tfr2 demonstrated the importance of hepatic TFR2 in iron metabolism [13], as they develop similar iron overload as the Tfr2 total knockout (KO) mice.…”

Transferrin Receptor 2: Evidence for Ligand-induced Stabilization and Redirection to a Recycling Pathway

“…The analogous mutation or knockout of Trfr2 in mice reproduces the disease phenotype (Fleming et al, 2002;Wallace et al, 2005). Thus, TfR2 is required for normal iron homeostasis.…”

“…TfR2 binds Tf in a pH-dependent manner, but its affinity for Fe 2 Tf (K D ϳ30 nM; Kawabata et al, 2000;West et al, 2000) is significantly lower than that of TfR1 (K D ϳ1 nM, Tsunoo and Sussman, 1983;Enns et al, 1991;Richardson and Ponka, 1997). Unlike TfR1, TfR2 expression is limited predominantly to hepatocytes (Kawabata et al, 1999;Fleming et al, 2000Fleming et al, , 2002Vogt et al, 2003;Calzolari et al, 2004;Zhang et al, 2004) and is not regulated by intracellular iron (Fleming et al, 2000;Kawabata et al, 2000Kawabata et al, , 2001. TfR2 cannot compensate for TfR1, whose knockout in mice results in embryonic lethality due to severe anemia (Levy et al, 1999).…”