This function is difficult to disclose when TFR2 is mutated in hemochromatosis type 3, because high transferrin saturation stabilizes TFR2 on plasma membrane and excess iron modifies erythropoiesis. as and are highly expressed in the spleen and in isolated erythroblasts from mice. Low hepcidin expression in is accounted for by erythroid expansion and production of the erythroid regulator erythroferrone. We suggest that Tfr2 is a component of a novel iron-sensing mechanism that adjusts erythrocyte production according to iron availability, likely by modulating the erythroblast Epo sensitivity. Introduction Transferrin receptor 2 (TFR2), the gene mutated in hemochromatosis type 31 is a transmembrane protein homologous to TFR1. Though not involved in iron transport, TFR2 binds the iron-loaded transferrin (holo-TF), even if with a lower NB-598 Maleate affinity than TFR1,2,3 a finding that Ntn1 suggests a potential regulatory role. TFR2 is expressed in the liver and, to a lower extent, in erythroid cells.2,4 In iron-replete conditions, TFR2 protein is stabilized on the plasma membrane by binding to its ligand holo-TF. This induces a reduction of TFR2 lysosomal degradation5 or a decreased shedding of the receptor from the plasma membrane (A.P., L.S., and C.C., unpublished manuscript). All of these properties make TFR2 a good candidate sensor for iron bound to circulating TF, measured as transferrin saturation (TS). Humans with mutations of develop iron overload1,6,7 with low hepcidin levels8; a similar phenotype occurs in mice with constitutive9-12 or liver conditional12,13 deletion. The hepatic form of TFR2 is proposed to cooperate with the hereditary hemochromatosis protein HFE, the atypical major histocompatibility complex class I protein, responsible for hemochromatosis type 1.14 The TFR2/HFE complex is presumed to activate the transcription of hepcidin (has been extensively studied, the erythroid function of the protein has not been investigated in depth. TFR2 and the erythropoietin receptor (EPOR) are activated synchronously and coexpressed during erythroid differentiation.2,16,17 Moreover, in erythroid precursors, TFR2 associates with EPOR in the endoplasmic reticulum and is required for the efficient transport of the receptor to the cell surface. Finally, knockdown in vitro delays the terminal differentiation of human erythroid progenitors.17 Thus, the erythroid NB-598 Maleate TFR2 is a component of the EPOR complex NB-598 Maleate and is required for efficient erythropoiesis. We have recently demonstrated that the phenotype of total (and liver-specific (knockout (KO) mice lacking the hepcidin inhibitor switches from iron overload to iron deficiency, overlapping the phenotype of mice. An intriguing finding in the double KO mice that we generated was that only mice developed erythrocytosis; this was not observed in mice.18 We hypothesized that this abnormality was accounted for by the loss of the erythroid Tfr2 in mice have lower hepcidin than and animals with liver-specific deletion of deletion rather than iron deficiency or variable hepcidin levels explain the observed phenotype. To unambiguously elucidate the function of TFR2 in erythropoiesis, particularly when iron-restricted, we generated a mouse model lacking in the erythroid precursors by NB-598 Maleate transplanting lethally irradiated wild-type (WT) mice with the bone marrow from donors and manipulated the dietary iron content of the transplanted animals. This model straightforwardly indicates that erythroid Tfr2 is essential to balance the red cell number according to the available iron, a crucial mechanism of adaptation to iron deficiency. Methods Mouse strains and bone marrow transplantation mice (129S2 strain) were as previously described.12 Bone marrow (BM) cells were harvested from 12 weeks old female mice or control WT littermates. Five 106 cells/mouse were injected IV into lethally irradiated (950 cGy) 8-week-old C57BL/6-Ly-5.1 male mice (Charles River). The animals were maintained in the animal facility of San Raffaele Scientific Institute (Milano, Italy) NB-598 Maleate in accordance with the European Union guidelines. The study was approved by the Institutional Animal Care and Use Committee of the San Raffaele Scientific Institute. Two months after BM transplantation (BMT), blood was collected by tail vein puncture into tubes containing 40 mg/mL EDTA for the evaluation of hematological parameters and donor/host chimerism. Mice were fed a standard diet (200 mg/kg carbonyl-iron, Scientific Animal Food and Engineering, SAFE, Augy, France) or an iron-deficient (ID) diet (iron content: 3.