Iron availability for erythropoiesis and its dysregulation in β-thalassemia are incompletely understood. We previously demonstrated that exogenous apotransferrin leads to more effective erythropoiesis, decreasing erythroferrone (ERFE) and derepressing hepcidin in β-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apotransferrin's effect is mediated via decreased TfR1 expression and evaluate TfR1 expression in β-thalassemic mice in vivo and in vitro with and without added apotransferrin. Our findings demonstrate that β-thalassemic erythroid precursors overexpress TfR1, an effect that can be reversed by the administration of exogenous apotransferrin. In vitro experiments demonstrate that apotransferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective β-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in β-thalassemic mice. To evaluate further, we crossed TfR1 mice, themselves exhibiting iron-restricted erythropoiesis with increased hepcidin, with β-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin derepression, and increased erythroid enucleation in relation to β-thalassemic mice. Our data demonstrate for the first time that TfR1 haploinsufficiency reverses iron overload specifically in β-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during β-thalassemic erythropoiesis, either directly via induced haploinsufficiency or via exogenous apotransferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron-restricted erythropoiesis and preventing systemic iron overload in β-thalassemic mice.
Iron overload results in significant morbidity and mortality in β-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in β-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb th1/th1 (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.
Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as β-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in β-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in β-thalassemia. We hypothesize that compensatory mechanisms are required in β-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2—a STAT5-dependent lipid binding protein downstream of erythropoietin—as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in β-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during β-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in β-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in β-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in β-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in β-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis.
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. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.
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