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

Abstract

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.

Figure 1. Non-haem iron concentrations in the liver (A) and spleen (B) of Tfr2 mutant (Tfr2 mut), non-iron-loaded (WT) and iron-loaded (WT + Fe) wild-type mice

Results are expressed as mean ± SEM, n = 6–8. Significant difference between mouse groups: * p <0.0001, Tfr2 mut vs WT, ⁺p <0.0001, Tfr2 mut vs WT + Fe and WT; ^#p <0.002 WT + Fe vs WT.

Figure 2. Plasma transferrin-bound iron uptake by the liver (A), spleen (B), duodenum (C) and kidneys (D) and plasma iron turnover (E) in Tfr2 mutant (Tfr2 mut), non-iron-loaded (WT) and iron-loaded (WT + Fe) wild-type mice

Results are expressed as mean ± SEM, n = 5–6. Significant difference between mouse groups: *p <0.05, Tfr2 mut vs WT + Fe; ⁺p <0.05, Tfr2 mut vs WT+Fe and WT; ^#p <0.01 WT + Fe vs WT.

Figure 3. Tfr2 protein expression in the liver (A) and spleen (B) in Tfr2 mutant (Tfr2 mut), non-iron-loaded (WT) and iron-loaded wild-type mice (WT + Fe)

Results are expressed as mean ± SEM, n = 5–6. Representative blots of hepatic (A) and splenic (B) Tfr2 protein expression from 3 independent experiments are shown. Significant difference between mouse groups: ⁺p <0.001, Tfr2 mut vs WT + Fe and WT; ^#p <0.05 WT + Fe vs WT.

Figure 4. Tfr1 protein expression in the liver (A) and spleen (B) in Tfr2 mutant (Tfr2 mut), non-iron-loaded (WT) and iron-loaded wild-type mice (WT + Fe)

Results are expressed as mean ± SEM, n = 4–6. Representative blots of hepatic and splenic Tfr1 protein expression from 3 independent experiments are shown. Significant difference between mouse groups: *p <0.001, Tfr2 mut vs WT; ⁺p <0.025, Tfr2 mut vs WT + Fe and WT; ^#p <0.025 WT + Fe vs WT.

Figure 5. Fpn protein expression in the liver (A) and spleen (B) in Tfr2 mutant (Tfr2 mut), non-iron-loaded (WT) and iron-loaded wild-type mice (WT + Fe)

Results are expressed as mean ± SEM, n = 4–7. Representative blots of hepatic and splenic Fpn protein expression from 3 independent experiments are shown. Significant difference between mouse groups: ⁺p <0.001, Tfr2 mut vs WT + Fe and WT; ^#p <0.001 WT + Fe vs WT.

Figure 6. Cellular localisation of hepatic Fpn expression. Frozen liver sections from wild-type (WT), Tfr2 mutant (Tfr2 mut) and iron-loaded wild-type mice (WT + Fe) were immunofluorescently stained with an antibody against Fpn (green) and DAPI (blue) for nuclear quantitation (A–C)

Double staining of Tfr2 mut liver for F4/80 (red, D) and Fpn (green, E) demonstrated that the majority of cells that strongly express Fpn were F4/80-positive macrophages (Mac), while hepatocytes (Hep) only express the Fpn antigen at lower levels (F).