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.
Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for erythropoiesis, and TfR1 is found in highest concentrations on erythroid precursors due to high iron requirement for hemoglobin (Hb) synthesis. Diseases of ineffective erythropoiesis such as β-thalassemia, are characterized by anemia, expanded and extramedullary erythropoiesis, and iron overload. Iron overload results from insufficient hepcidin, a peptide hormone secreted by hepatocytes in response to iron load. In β-thalassemia, hepcidin is relatively suppressed as a consequence of erythroid expansion. Erythroferrone (ERFE), a recently described erythroid-derived hepcidin suppressor, has been proposed as the mechanism and found in higher concentration in bone marrow of β-thalassemic mice. We previous demonstrate that exogenous transferrin (Tf) ameliorates anemia in β-thalassemic mice, reversing splenomegaly, hepcidin suppression, and iron overload and recently confirmed a decrease in Erfe expression in erythroid precursors from Tf-treated β-thalassemic mice. We observed that although Tf-treated β-thalassemic mice exhibit a further decrease in MCV and MCH, suggesting a relatively more iron restricted erythropoiesis, TfR1 expression is decreased. We hypothesize that TfR1 is central to Tf's effect on erythropoiesis in β-thalassemic mice. Last year, we presented our analysis of th3/+ TfR1+/- double heterozygote mice which exhibit reversal of all erythropoiesis- and iron-related pathology in th3/+ mice, confirming our observations in Tf-treated β-thalassemic mice and further supporting our hypothesis. To evaluate the mechanism involved, we observed that despite suppressed TfR1 concentration in reticulocyte (P=0.006) and sorted bone marrow erythroid precursors (P=0.0004) from Tf-treated th1/th1 mice, cell surface TfR1 expression decreased on reticulocytes (P=0.003) but was surprisingly increased on late stage erythroid precursors (P=0.007) (Figure 1A), suggesting that exogenous Tf influences erythroid precursor enucleation. Because we previously demonstrate decreased serum soluble TfR1 in Tf-treated th1/th1 mice [Liu J Blood 2013], we hypothesize that exogenous Tf alters TfR1 shedding from erythroid precursor membranes, promoting enucleation and improved terminal differentiation. We observed decreased enucleation using syto60 in flow cytometry of fetal liver cells (FLC) from th3/+ relative to wild type (WT) embryos (35 vs. 51%, P=0.03) which is normalized by exposure of th3/+ FLCs to Tf in vitro (58 vs. 41%, P=0.001) (Figure 1B). Tf-treated th3/+ FLCs shed more TfR1 to the nuclear fraction relative to reticulocyte during enucleation (P=0.0001) (Figure 1C). Furthermore, enucleation isdecreased in vivo in th3/+ relative to WT FLCs and peripheral blood at E14.5 and normalized in th3/+ TfR1+/- double heterozygote mice (45 vs. 35%, P=0.002) (Figure 1D). Interestingly, we analyzed iron status in TfR1+/- mice revealing that serum hepcidin is increased relative to WT (323 vs. 190 ng/ml, P=0.04) despite minimally decreased serum and liver iron concentrations (no statistically significant differences) and increased Erfe expression in erythroid precursors (5-fold, P=0.04). Relative to th3/+ mice, double heterozygote mice exhibit decreased serum iron (94 vs. 133 ug/dl), non-heme liver iron (0.31 vs. 0.74 mg iron/g dry weight, P=0.02), and Erfe expression (0.3-fold, P=0.04). Although no difference is observed between double heterozygote mice and th3/+, serum hepcidin is significantly increased in double heterozygote mice compare to WT (392 vs. 190 ng/ml, P=0.01), suggesting a more appropriate hepcidin response to iron overload (Figure 1E). Taken together, we postulate that decreased TfR1 expression plays a critical role in reversing ineffective erythropoiesis by increasing enucleation and influences hepcidin regulation in an ERFE independent manner. Disclosures No relevant conflicts of interest to declare.
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.
β-thalassemia is an inherited blood disorder caused by reduced or absence of β-globin expression which results in imbalanced globin synthesis, ineffective erythropoiesis, and anemia. How the imbalance between α- and β-globin results in ineffective erythropoiesis, if apoptosis or dysfunctional differentiation of erythroid precursors results in ineffective erythropoiesis, and whether disrupted iron regulation and / or iron overload in β-thalassemia is directly involved in the pathophysiology of ineffective erythropoiesis is incompletely understood. Iron is critical for hemoglobin synthesis and erythropoiesis is dependent on transferrin (Tf) bound iron. Tf functions as the main iron transporter in circulation, where it exists in three forms: as iron-free apo-transferrin (apoTf), monoferric Tf, or diferric Tf (holoTf). Typically, iron is bound to 30% of all Tf binding sites in circulation at which point monoferric Tf is found in the highest concentration relative to holoTf. . We have previously shown that exogenous apoTf ameliorates anemia in a mouse model of β-thalassemia intermedia (th1/th1), resulting in reduced splenomegaly, reticulocytosis, and α-globin precipitation on circulatory red blood cells (RBC). We also observe a decrease in mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV), serum iron, Tf saturation, together suggesting that relative iron deficiency improves iron metabolism and ineffective erythropoiesis in apoTf-treated th1/th1 mice. We hypothesize that exogenous apoTf decreases cytosolic iron and heme as a consequence of increased monoferric Tf which results in less iron entering cells via Tf:TfR1 binding. Our current data reveals that in vitro incubation of purified apoTf and holoTf at 37C results in the formation of monoferric Tf, and injection of wild-type (WT) mice with a single intraperitoneal dose of apoTf (10mg) decreases holoTf (P=0.01) and increases monoferric Tf (P=0.02) in the serum 6 hours after injection. Using both calcium mobilization and anti-Tf antibodies in flow cytometry, we demonstrate that apoTf results in no TfR1 binding relative to holoTf in CHO cells (P<0.0001), and an equal concentration apoTf:holoTf mixture results in intermediate binding between apoTf and holoTf (P=0.004). We also demonstrate a dose dependent decrease in cytosolic iron (P<0.001) and heme (P<0.01) in MEL cells treated with escalating concentrations of apoTf, an effect abrogated by the additional of DFO, an iron chelator; urea gel of MEL cell supernatants reveals that monoferric Tf increases with increasing doses of apoTf and decreased in concurrently DFO treated cells (Figure 1). These findings together suggest that changes in iron uptake result from increased monoferric Tf (rather than competition of apoTf for TfR1 binding sites). In addition, in vivo experiments demonstrate a decrease in heme concentration (56 vs. 67 uM, P<0.0001) in circulating RBCs as well as α-globin (1.3 vs. 3.3-fold, P=0.009) and β-globin (Figure 2) mRNA expression in sorted bone marrow orthochromatophilic erythroblasts from apoTf- vs. PBS-treated th1/th1 mice. As heme is known to regulate globin expression through transcriptional and translational routes, we evaluate bach1, heme oxygenase 1 (HO-1), and heme-regulated eIF2α kinase (HRI). We demonstrate no difference in bach1 and HO-1 mRNA expression but a significant and unexpected decrease in HRI expression (1.0 vs. 2.8-fold, P=0.01) in sorted bone marrow orthochromatophilic erythroblasts from apoTf- vs. PBS-treated th1/th1 mice (similar findings in all stages of terminal erythroid differentiation), suggesting that HRI is regulated by mechanisms independent of cellular iron and heme. Furthermore, western blots of HRI and its target eIF2α (total and phosphorylated) are also normalized in sorted bone marrow erythroid precursors from apoTf- vs. PBS-treated th1/th1 mice (Figure 3). Taken together, our data suggests that exogenous apoTf in th1/th1 mice results in decreased cytosolic iron and heme as well as α- and β-globin synthesis as a consequence of increased monoferric Tf in circulation and provides a novel system to explore the regulation of globin in β-thalassemia and how iron deficiency benefits this disease. Disclosures No relevant conflicts of interest to declare.
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