Transferrin Receptor 2 in iron metabolism: its role in the liver and extra-hepatic tissue

Abstract

Transferrin receptor 2 (TFR2) is an important regulator of systemic iron metabolism. Patients with TFR2 mutations develop type III hereditary haemochromatosis (HH), which is associated with inappropriate mRNA levels of the iron regulatory hormone hepcidin (HAMP). Studies from our laboratory using mice with hepatocyte-specific deletion of TfR2 suggested that hepatic TFR2 is important in the regulation of body iron homeostasis. How TFR2 regulates Hamp and thus iron homeostasis is still unclear, with two main schools of thought. According to one, when body iron levels increase, TFR2 interacts with HFE, the haemochromatosis protein, which leads to a signalling cascade and increase in HAMP transcription. The second states that HFE and TFR2 can act independently of each other to regulate HAMP. Although highly expressed in the liver, TfR2 expression has also been reported in erythroid cells, macrophages, peripheral blood mononuclear cells, spleen, testes and prostate gland. The role of TFR2 in these tissues and cells is not known. Some studies have suggested that TFR2 could have roles independent of iron regulation in the spleen and brain. Genome wide association studies have shown a strong association between TFR2 and haematological parameters. Recent publications have also suggested a role for TfR2 in erythropoiesis. The objective of this thesis was to increase our understanding of the biology of TFR2 and its role in various tissues. This thesis examines the role that TFR2 plays in iron homeostasis, inflammation-mediated iron homeostasis and erythropoiesis by investigating various tissue-specific TfR2 knockout mice. In order to examine the putative interaction between HFE and TFR2, a novel stable co-expression model system was used. HFE and TFR2 were expressed under the same promoter using a novel tricistronic vector. Protein-protein interactions were studied in vivo in the native state of the proteins using the proximity ligation assay and by co-immunoprecipitation. Results of these studies indicate that HFE and TFR2 do not interact and that they can independently regulate Hamp. In order to understand the hepatic function of TfR2, TfR2-/- mice and mice with a hepatocyte-specific deletion of TfR2 (TfR2f/f/Alb-Cre+/-) were analysed. Results from these studies suggest that hepatic TFR2 may be involved in appropriate regulation of bone morphogenetic protein (Bmp) signalling in response to increased iron. The loss of TFR2 in hepatocytes leads to reduced BMP signalling. The role of TfR2 in cells of the monocyte/macrophage lineage was examined by generating mice lacking TfR2 in these cells. TfR2f/f/LysM-Cre+/- mice were generated by crossing TfR2f/f with LysM-Cre+/- mice. In order to examine the role of macrophage TFR2 in iron-regulated and inflammation-regulated iron homeostasis the mice were either fed an iron-rich diet or injected with lipopolysaccharide (LPS). Analysis of genes involved in the regulation of iron metabolism in the liver and spleen of TfR2f/f/LysM-Cre+/- mice indicates that macrophage TfR2 does not play a role in regulating systemic iron metabolism in response to increased body iron levels or inflammation. The expression level of the iron exporter ferroportin 1 (Fpn1) was lower in the spleen and the livers of TfR2f/f/LysM-Cre+/- mice suggesting that macrophage expression of TfR2 may be required for transcriptional regulation of Fpn1. Mice treated with LPS had significantly reduced expression of all the major genes thought to be involved in the iron regulatory pathway. Previous studies have shown a reduction in the levels Fpn1 and hemojuvelin (Hjv) in the liver; the studies in this thesis reveal that the impact of inflammatory stimuli on the transcriptional regulation of iron metabolism related genes may be broader than previously reported. The role of TfR2 in normal and stress erythropoiesis was examined by generating mice with an erythroid-specific deletion of TfR2. This was achieved by crossing the TfR2D/f mice with Vav-Cre+/-. These mice were then fed either an iron-rich or an iron-deficient diet to analyse the effect of stress on the erythroid function of TFR2. Mice lacking TfR2 in erythroid cells had significant splenomegaly and extramedullary haematopoiesis as compared to control anaemic animals. Analysis of the developmental stages of erythropoiesis revealed that the lack of TfR2 in erythroid cells leads to a developmental block at the polychromatic erythroblast stage. The findings of this thesis have led to a greater understanding of the biology and importance of TFR2 in different tissues. The results described in Chapters 3 and 4 contribute to our understanding of the role played by TFR2 in the hepatocyte and regulation of iron metabolism in general. The results in the fifth chapter reveal an as yet unexplored link between inflammation and BMP-SMAD mediated regulation of iron metabolism. The sixth chapter reveals an important role of erythroid TFR2 in the differentiation of erythroblasts in stress conditions. It also supports the paradigm that proteins expressed at lower levels in some tissues could have tissue-specific functions in addition to their predominant role in the main tissue where they are highly expressed.