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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Background & Aims HFE and transferrin receptor 2 (TFR2) are each necessary for the normal relationship between body iron status and liver hepcidin expression. In murine Hfe and Tfr2 knockout models of hereditary hemochromatosis (HH), signal transduction to hepcidin via the bone morphogenetic protein 6 (Bmp6)/Smad1,5,8 pathway is attenuated. We examined the effect of dietary iron on regulation of hepcidin expression via the Bmp6/Smad1,5,8 pathway using mice with targeted disruption of Tfr2, Hfe, or both genes. Methods Hepatic iron concentrations and mRNA expression of Bmp6 and hepcidin were compared with wild-type mice in each of the HH models on standard or iron-loading diets. Liver phospho-Smad (P-Smad)1,5,8 and Id1 mRNA levels were measured as markers of Bmp/Smad signaling. Results While Bmp6 expression was increased, liver hepcidin and Id1 expression were decreased in each of the HH models compared with wild-type mice. Each of the HH models also demonstrated attenuated P-Smad1,5,8 levels relative to liver iron status. Mice with combined Hfe/Tfr2 disruption were most affected. Dietary iron loading increased hepcidin and Id1 expression in each of the HH models. Compared with wild-type mice, HH mice demonstrated attenuated (Hfe knockout) or no increases in P-Smad1,5,8 levels in response to dietary iron loading. Conclusions These observations demonstrate that Tfr2 and Hfe are each required for normal signaling of iron status to hepcidin via Bmp6/Smad1,5,8 pathway. Mice with combined loss of Hfe and Tfr2 up-regulate hepcidin in response to dietary iron loading without increases in liver BMP6 mRNA or steady-state P-Smad1,5,8 levels.

A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitochondria isolated from transfected COS cells. Northern blot analyses of total RNAs from mouse tissues and Western blot analyses of mouse tissue homogenates showed differences in tissue-specific expression between CA VB and CA VA. CA VB was readily detected in most tissues, while CA VA expression was limited to liver, skeletal muscle, and kidney. The human orthologue of murine CA VB was recently reported also. Comparison of the CA domain sequence of human CA VB with that reported here shows that the CA domains of CA VB are much more highly conserved between mouse and human (95% identity) than the CA domains of mouse and human CA VAs (78% identity). Analysis of phylogenetic relationships between these and other available human and mouse CA isozyme sequences revealed that mammalian CA VB evolved much more slowly than CA VA, accepting amino acid substitutions at least 4.5 times more slowly since each evolved from its respective humanmouse ancestral gene around 90 million years ago. Both the differences in tissue distribution and the much greater evolutionary constraints on CA VB sequences suggest that CA VB and CA VA have evolved to assume different physiological roles.

Hepcidin is a hepatocellular hormone that inhibits the release of iron from certain cell populations, including enterocytes and reticuloendothelial cells. The regulation of hepcidin ( HAMP) gene expression by iron status is mediated in part by the signaling molecule bone morphogenetic protein 6 (BMP6). We took advantage of the low iron status of juvenile mice to characterize the regulation of Bmp6 and Hamp1 expression by iron administered in three forms: 1) ferri-transferrin (Fe-Tf), 2) ferric ammonium citrate (FAC), and 3) liver ferritin. Each of these forms of iron enters cells by distinct mechanisms and chemical forms. Iron was parenterally administered to 10-day-old mice, and hepatic expression of Bmp6 and Hamp1 mRNAs was measured 6 h later. We observed that hepatic Bmp6 expression increased in response to ferritin but was unchanged by Fe-Tf or FAC. Hepatic Hamp1 expression likewise increased in response to ferritin and Fe-Tf but was decreased by FAC. Exogenous ferritin increased Bmp6 and Hamp1 expression in older mice as well. Removing iron from ferritin markedly decreased its effect on Bmp6 expression. Exogenously administered ferritin and the derived iron localized in the liver primarily to sinusoidal lining cells. Moreover, expression of Bmp6 mRNA in isolated adult rodent liver cells was much higher in sinusoidal lining cells than hepatocytes (endothelial >> stellate > Kupffer). We conclude that exogenous iron-containing ferritin upregulates hepatic Bmp6 expression, and we speculate that liver ferritin contributes to regulation of Bmp6 and, thus, Hamp1 genes.

The inhibitory Smad7 acts as a critical suppressor of hepcidin, the major regulator of systemic iron homeostasis. In this study we define the mRNA expression of the two functionally related Smad proteins, Smad6 and Smad7, within pathways known to regulate hepcidin levels. Using mouse models for hereditary hemochromatosis (Hfe-, TfR2-, Hfe/TfR2-, Hjv- and hepcidin1-deficient mice) we show that hepcidin, Smad6 and Smad7 mRNA expression is coordinated in such a way that it correlates with the activity of the Bmp/Smad signaling pathway rather than with liver iron levels. This regulatory circuitry is disconnected by iron treatment of Hfe-/- and Hfe/TfR2 mice that significantly increases hepatic iron levels as well as hepcidin, Smad6 and Smad7 mRNA expression but fails to augment pSmad1/5/8 levels. This suggests that additional pathways contribute to the regulation of hepcidin, Smad6 and Smad7 under these conditions which do not require Hfe.