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...

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. Liver disease resulting from iron toxicity is the major cause of death in HH. Hepatic iron loading in HH is progressive despite downregulation of the classical transferrin receptor (TfR). Recently a human cDNA highly homologous to TfR was identified and reported to encode a protein (TfR2) that binds holotransferrin and mediates uptake of transferrin-bound iron. We independently identified a full-length murine EST encoding the mouse orthologue of the human TfR2. Although homologous to murine TfR in the coding region, the TfR2 transcript does not contain the ironresponsive elements found in the 3 untranslated sequence of TfR mRNA. To determine the potential role for TfR2 in iron uptake by liver, we investigated TfR and TfR2 expression in normal mice and murine models of dietary iron overload (2% carbonyl iron), dietary iron deficiency (gastric parietal cell ablation), and HH (HFE ؊͞؊).Northern blot analyses demonstrated distinct tissue-specific patterns of expression for TfR and TfR2, with TfR2 expressed highly only in liver where TfR expression is low. In situ hybridization demonstrated abundant TfR2 expression in hepatocytes. In contrast to TfR, TfR2 expression in liver was not increased in iron deficiency. Furthermore, hepatic expression of TfR2 was not downregulated with dietary iron loading or in the HFE ؊͞؊ model of HH. From these observations, we propose that TfR2 allows continued uptake of Tf-bound iron by hepatocytes even after TfR has been down-regulated by iron overload, and this uptake contributes to the susceptibility of liver to iron loading in HH.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by tissue iron deposition secondary to excessive dietary iron absorption. We recently reported that HFE, the protein defective in HH, was physically associated with the transferrin receptor (TfR) in duodenal crypt cells and proposed that mutations in HFE attenuate the uptake of transferrin-bound iron from plasma by duodenal crypt cells, leading to up-regulation of transporters for dietary iron. Here, we tested the hypothesis that HFE ؊/؊ mice have increased duodenal expression of the divalent metal transporter (DMT1). Hereditary hemochromatosis (HH) is a common disorder of iron homeostasis in which the intestinal absorption of iron is excessive in relation to body iron status (1-5). The excess iron is deposited in the parenchyma of many tissues, leading to tissue damage and organ failure. Clinical consequences include liver cirrhosis, hepatocellular carcinoma, diabetes, heart failure, arthritis, and hypogonadism (6-9). The gene defective in HH, designated HFE, was found to encode a major histocompatibility complex (MHC) class I-like integral membrane protein (10) that had no obvious relationship to iron absorption. A link between HFE and iron metabolism was provided by the observations in human placenta that the HFE protein is localized on the apical surface of syncytiotrophoblast cells (the site of transferrin-mediated maternal-fetal iron transport) and is physically associated with the transferrin receptor (TfR) (11). Physical association between TfR and expressed recombinant HFE protein was also reported in cultured cells (12, 13) and in vitro (14). In cell culture, overexpressed recombinant HFE (but not HFE carrying the HH mutation) was reported to reduce the affinity of TfR for holotransferrin, suggesting a role for normal HFE in down-regulating transferrin-mediated iron uptake. Although loss of this downregulation of transferrin-mediated iron transport might explain the excess deposition of tissue iron in HH patients, it would not explain the excess absorption of dietary iron (15). Studies on HH patients suggest that the primary defect is loss of the normal feedback mechanisms regulating absorption of dietary (nontransferrin-bound) iron across the intestinal mucosa (2-5).Dietary iron absorption is normally tightly linked with body utilization through the sensing of body iron status in the proximal small intestine (16,17). Several lines of evidence indicate that the body iron status is detected by the uptake of transferrin-bound iron from plasma at the basolateral surface of intestinal crypt cells (18,19). We recently demonstrated that HFE colocalizes with and is physically associated with TfR in these cells (1). This observation suggests a mechanism by which HFE might participate in sensing body iron status by modulating transferrin-mediated uptake of plasma iron in the crypt cells. We propose that mutations of HFE in HH patients impair transferrin-mediated iron uptake and thus decrease crypt cell uptake of plasm...

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