Increased Transferrin Concentration Ameliorates Anemia in Beta-Thalassemic Mice Through Changes in Iron Uptake and TfR1 Trafficking

Abstract

906 Hemoglobin (Hb) synthesis during terminal erythroid differentiation is iron dependent and iron delivery requires transferrin (Tf) to transferrin receptor 1 (TfR1) binding. After binding of Tf to TfR1, the ligand-receptor complex is endocytosed through a clathrin dependent mechanism, results in iron delivery via the endosomal compartment, and ends with TfR1 recycling back to the plasma membrane. During erythroid differentiation, TfR1 expression is downregulated and is completely absent from the mature red blood cells (RBCs). Reticulocyte TfR1 is rerouted from the endosomal recycling pathway by sorting to exosomes where it is shed from the cell. Exosomes are small membrane vesicles originating from the fusion of a multi-vesicular endosome compartment with the plasma membrane, leading to the secretion of intraluminal vesicles into circulation. Cell surface TfR1 expression is proportional to the concentration of soluble TfR1 found in circulation as a consequence of exosomal secretion and is increased both in iron deficiency and expanded erythropoiesis. Increased soluble TfR1 is observed in disease of expanded and ineffective erythropoiesis (IE) such as beta-thalassemia, a disease associated with anemia, extramedullary hematopoiesis (EMH), and splenomegaly. We previously demonstrated that apoTf-treated beta-thalassemic mice have more circulating RBCs, increased Hb, reversed splenomegaly and EMH, with fewer reticulocytes and erythroid precursors in the bone marrow and spleen. Furthermore, in both beta-thalassemic and C57BL/6 mice treated with apoTf, we observed a significant reduction in mean cellular hemoglobin (MCH). We hypothesize that TfR1 trafficking is impaired in beta-thalassemia and that exogenous apoTf reduces cellular iron uptake and normalizes TfR1 trafficking pathways, resulting in reduced heme synthesis and a lower MCH observed in apoTf-treated mice. To test this hypothesis, we evaluate 1) cellular iron uptake in cell culture and 2) TfR1 mRNA expression, cell surface expression, and endosomal/exosomal trafficking pathways in C57BL/6 and beta-thalassemic mice. Cell culture experiments were performed using K562 cells +/− 50- and 250-fold excess apoTf relative to holoTf. Mice were evaluated after 20 days of 10 mg human apoTf IP injections (compared with PBS injection). Using a calcein fluorescence quenching approach, we demonstrate that exogenous apoTf decreases iron uptake in culture in a dose response manner. Furthermore, in mouse bone marrow samples sorted using CD44/TER119, we show that TfR1 mRNA expression is higher in beta-thalassemic relative to C57BL/6 mice and increases further in apoTf treated mice in all stages of terminal erythroid differentiation. In addition, although western blot experiments show an increase in cellular TfR1 in beta-thalassemic relative to C57BL/6 mice, cell fractionation experiments demonstrate a proportional shift from the plasma membrane to the endosomal compartment in apoTf-treated mice and reticulocytes of apoTf treated beta-thalassemic mice exhibit significantly reduced TfR1 expression per cell. Finally, reticulocyte TfR1 sorting into exosomes is impaired in beta-thalassemic relative to C57BL/6 mice with a proportional increase in exosomal TfR1 clearance and a reduction in soluble TfR1 in the serum in apoTf treated beta-thalassemic mice. Taken together, our findings demonstrate for the first time that TfR1 trafficking is important for RBC physiology separately from its role in iron uptake and that exogenous apoTf enhances TfR1 endosomal trafficking, normalizes TfR1 sorting into exosomes, and reduces cellular iron uptake. Finally, our results elucidate mechanisms by which MCH is reduced in apoTf-treated mice and provide evidence for multiple consequences of Tf:TfR1 binding on erythroid differentiation and proliferation that characterize diseases of IE. Disclosures: No relevant conflicts of interest to declare.