Changes in TfR1 Expression and Trafficking Correlate with Erythroid Effectiveness in β-Thalassemic Mice

Abstract

Transferrin receptor 1 (TfR1) is found in highest concentrations on erythroid precursors due to the disproportionately high iron requirement for hemoglobin synthesis, making transferrin-bound iron binding to TfR1 essential for erythropoiesis. Recent data reveals that TfR1 mRNA expression (6.48±2.23 vs. 1.0±0.25 relative to GAPDH, P=0.04 in sorted basophilic erythroblasts), whole cell protein concentration measured using ImageJ (11496±1783 vs. 1620±1448, P=0.0001 in reticulocytes), and cell surface concentration measured using flow cytometry (mean fluorescence index 17314±2370 vs. 11930±2530, P=0.002 in bone marrow basophilic erythroblasts) are increased in β-thalassemic (th1/th1) relative to wild type (WT) mice. We hypothesized that a relative decrease in TfR1 expression would improve the phenotype in β-thalassemic mice and crossed TfR1+/- (TfR1 heterozygote) mice [Levy JE Nat Gen 1999] with th3/+ mice, another commonly used mouse model of β-thalassemia. Of the 50 pups born, 13 had th3 genotype, 12 (92%) of which also contained the mutant TfR1, suggesting a strong survival advantage of TfR1 heterozygote th3/+ (compound heterozygotes) relative to th3/+ mice. Analysis of 3-4 month old compound heterozygotes revealed a significant decrease in splenomegaly (0.007±0.001 vs. 0.016±0.0041 g spleen/g body weight, P=0.0009), reticulocytosis (1019±186 vs. 1672±218 x 10^9 cells/uL, P=0.001), and α-globin precipitation on circulating RBCs (Figure 1) relative to th3/+ mice. Furthermore, compound heterozygotes exhibit improvement in circulating RBCs (12±0.1 vs. 9±0.6 x 10^6 cells/uL, P<0.0001) and hemoglobin (10±0.3 vs. 8.2±0.3 g/dL, P=0.0004) and decrease in MCH (8.9±0.2 vs. 10±0.2 pg, P=0.002) and non-heme liver iron (0.31±0.14 vs. 0.74±0.29 mg iron/g dry weight, P=0.02) relative to th3/+ mice. These findings suggest that decreased TfR1 expression results in more efficient erythropoiesis in β-thalassemia.We previously demonstrate that exogenous apo-transferrin (apoTf) injections result in more circulating RBCs, increased hemoglobin, and reversal of splenomegaly in th1/th1 mice [Li H Nat Med 2010]. We hypothesize that ineffective erythropoiesis in th1/th1 mice is TfR1-mediated and involves excess iron delivery to erythroid precursors. To further explore the role of TfR1 in erythropoiesis, we evaluate apoTf-treated th1/th1 mice. TfR1 mRNA expression is unchanged in apoTf-treated relative to untreated th1/th1 mice despite more iron restricted erythropoiesis (MCH 24.56±0.72 vs. 33.98±1.67 pg, P<0.0001) and a significant decrease in serum soluble TfR1 [Liu J Blood 2013]. Western blots of reticulocytes from apoTf-treated th1/th1 mice reveal less TfR1 (4914±2561 vs. 11496±1783, P=0.006) and erythroid precursors from apoTf-treated th1/th1 mice analyzed by flow cytometry reveal more TfR1 (mean fluorescence index 24311±6025 vs. 11496±1783, P=0.02 in basophilic erythroblasts) relative to untreated th1/th1 mice. We hypothesized that TfR1 localization in sub-cellular compartments is altered in th1/th1 relative to WT mice and that increased apoTf enables normalization of TfR1 trafficking. Using differential centrifugation, we analyzed TfR1 in sub-cellular fractions in vivo and in vitro. Our results demonstrate a relative increase in membrane-associated and endosomal TfR1 in sorted bone marrow erythroid precursors from apoTf-treated relative to untreated th1/th1 mice. Furthermore, in vitro experiments also demonstrate increased membrane-associated and endosomal TfR1 in fetal liver cells from apoTf-treated relative to untreated th3/+ embryos (Figure 2). Lastly, we analyzed TfR1 exosomal release from reticulocytes after 2 days in culture, a commonly used method for exosome analysis, and demonstrate that exosomal release is decreased in reticulocytes from apoTf-treated relative to untreated th1/th1 mice (Figure 3).Taken together, our data suggest that TfR1 plays a critical role in erythropoiesis, both in an iron-dependent and possibly independent capacity. We postulate that a defect in TfR1 trafficking, perhaps with a delayed or incomplete removal of TfR1 during erythroid differentiation, occurs in β-thalassemia, that reduction of TfR1 in β-thalassemic mice partially reverses ineffective erythropoiesis, and that exogenous apoTf decreases TfR1 expression and exosomal release while increasing membrane and endosomal cycling. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.