Disruption of hemochromatosis protein and transferrin receptor 2 causes iron-induced liver injury in mice.

UNLABELLED: Mutations in hemochromatosis protein (HFE) or transferrin receptor 2 (TFR2) cause hereditary hemochromatosis (HH) by impeding production of the liver iron-regulatory hormone, hepcidin (HAMP). This study examined the effects of disruption of Hfe or Tfr2, either alone or together, on liver iron loading and injury in mouse models of HH. Iron status was determined in Hfe knockout (Hfe(-/-)), Tfr2 Y245X mutant (Tfr2(mut)), and double-mutant (Hfe(-/-) ×Tfr2(mut) ) mice by measuring plasma and liver iron levels. Plasma alanine transaminase (ALT) activity, liver histology, and collagen deposition were evaluated to assess liver injury. Hepatic oxidative stress was assessed by measuring superoxide dismutase (SOD) activity and F(2)-isoprostane levels. Gene expression was measured by real-time polymerase chain reaction. Hfe(-/-) ×Tfr2(mut) mice had elevated hepatic iron with a periportal distribution and increased plasma iron, transferrin saturation, and non-transferrin-bound iron, compared with Hfe(-/-), Tfr2(mut), and wild-type (WT) mice. Hamp1 expression was reduced to 40% (Hfe(-/-) and Tfr2(mut) ) and 1% (Hfe(-/-) ×Tfr2(mut)) of WT values. Hfe(-/-) ×Tfr2(mut) mice had elevated plasma ALT activity and mild hepatic inflammation with scattered aggregates of infiltrating inflammatory cluster of differentiation 45 (CD45)-positive cells. Increased hepatic hydoxyproline levels as well as Sirius red and Masson's Trichrome staining demonstrated advanced portal collagen deposition. Hfe(-/-) and Tfr2(mut) mice had less hepatic inflammation and collagen deposition. Liver F(2) -isoprostane levels were elevated, and copper/zinc and manganese SOD activities decreased in Hfe(-/-) ×Tfr2(mut), Tfr2(mut), and Hfe(-/-) mice, compared with WT mice. CONCLUSION: Disruption of both Hfe and Tfr2 caused more severe hepatic iron overload with more advanced lipid peroxidation, inflammation, and portal fibrosis than was observed with the disruption of either gene alone. The Hfe(-/-) ×Tfr2(mut) mouse model of iron-induced liver injury reflects the liver injury phenotype observed in human HH.

Brain transcriptome perturbations in the Hfe(-/-) mouse model of genetic iron loading.

Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients.

Hepatic iron loading in mice increases cholesterol biosynthesis.

UNLABELLED: Iron and cholesterol are both essential metabolites in mammalian systems, and too much or too little of either can have serious clinical consequences. In addition, both have been associated with steatosis and its progression, contributing, inter alia, to an increase in hepatic oxidative stress. The interaction between iron and cholesterol is unclear, with no consistent evidence emerging with respect to changes in plasma cholesterol on the basis of iron status. We sought to clarify the role of iron in lipid metabolism by studying the effects of iron status on hepatic cholesterol synthesis in mice with differing iron status. Transcripts of seven enzymes in the cholesterol biosynthesis pathway were significantly up-regulated with increasing hepatic iron (R(2) between 0.602 and 0.164), including those of the rate-limiting enzyme, 3-hydroxy-3-methylglutarate-coenzyme A reductase (Hmgcr; R(2) = 0.362, P < 0.002). Hepatic cholesterol content correlated positively with hepatic iron (R(2) = 0.255, P < 0.007). There was no significant relationship between plasma cholesterol and either hepatic cholesterol or iron (R(2) = 0.101 and 0.014, respectively). Hepatic iron did not correlate with a number of known regulators of cholesterol synthesis, including sterol-regulatory element binding factor 2 (Srebf2; R(2) = 0.015), suggesting that the increases seen in the cholesterol biosynthesis pathway are independent of Srebf2. Transcripts of genes involved in bile acid synthesis, transport, or regulation did not increase with increasing hepatic iron. CONCLUSION: This study suggests that hepatic iron loading increases liver cholesterol synthesis and provides a new and potentially important additional mechanism by which iron could contribute to the development of fatty liver disease or lipotoxicity.

Iron uptake from plasma transferrin by a transferrin receptor 2 mutant mouse model of haemochromatosis.

BACKGROUND & AIMS: Hereditary haemochromatosis type 3 is caused by mutations in transferrin receptor (TFR) 2. TFR2 has been shown to mediate iron transport in vitro and regulate iron homeostasis. The aim of this study was to determine the role of Tfr2 in iron transport in vivo using a Tfr2 mutant mouse. METHODS: Tfr2 mutant and wild-type mice were injected intravenously with (59)Fe-transferrin and tissue (59)Fe uptake was measured. Tfr1, Tfr2 and ferroportin expression was measured by real-time PCR and Western blot. Cellular localisation of ferroportin was determined by immunohistochemistry. RESULTS: Transferrin-bound iron uptake by the liver and spleen in Tfr2 mutant mice was reduced by 20% and 65%, respectively, whilst duodenal and renal uptake was unchanged compared with iron-loaded wild-type mice. In Tfr2 mutant mice, liver Tfr2 protein was absent, whilst ferroportin protein was increased in non-parenchymal cells and there was a low level of expression in hepatocytes. Tfr1 expression was unchanged compared with iron-loaded wild-type mice. Splenic Tfr2 protein expression was absent whilst Tfr1 and ferroportin protein expression was increased in Tfr2 mutant mice compared with iron-loaded wild-type mice. CONCLUSIONS: A small reduction in hepatic transferrin-bound iron uptake in Tfr2 mutant mice suggests that Tfr2 plays a minor role in liver iron transport and its primary role is to regulate iron metabolism. Increased ferroportin expression due to decreased hepcidin mRNA levels is likely to be responsible for impaired splenic iron uptake in Tfr2 mutant mice.

Transferrin receptor 2 mediates uptake of transferrin-bound and non-transferrin-bound iron.

BACKGROUND/AIMS: Transferrin receptor 2 appears to have dual roles in iron metabolism; one is signalling, the other is iron transport. It is sensitive to high levels of diferric transferrin, which is associated with disorders of iron overload. Also present in these disorders are increased levels of plasma non-transferrin-bound iron. This study sought to clarify the role of transferrin receptor 2 in the uptake of transferrin-bound and non-transferrin-bound iron. METHODS: Variant Chinese Hamster Ovary (CHO) cells, transfected with transferrin receptor 2, were incubated with radio-labelled transferrin-bound or non-transferrin-bound iron. Competition studies were performed in the presence of unlabelled dimetallic transferrin; knockdown was performed using specific siRNA. RESULTS: Cells expressing transferrin receptor 2 bound and internalised transferrin and transferrin-bound iron. Transferrin recycling occurred with an average cycling time of 11-15 min. Interestingly, the presence of transferrin receptor 2 was also associated with uptake of non-transferrin-bound iron which was inhibited by unlabelled transferrin-bound metals. Knockdown reduced transferrin-bound and non-transferrin-bound iron uptake by approximately 60%. CONCLUSIONS: Transferrin receptor 2 mediates transferrin-bound iron uptake by receptor-mediated endocytosis. It is also involved in the uptake of non-transferrin-bound iron and the inhibition of non-transferrin-bound iron uptake by diferric transferrin in CHO cells.