Another main interest of the laboratory is to understand the regulation of erythrocyte differentiation and survival. Epo binding to Epo receptor triggers a complicated and incompletely understood set of potentially related molecular signals influencing cell survival, differentiation, and enucleation. Although Epo is associated with increased survival of erythroid precursors, it induces reactive oxygen species (ROS), and high Epo concentration has an anti-enucleation effect in vitro [Zhao Exp Hematol 2016]. Furthermore, diseases of ineffective erythropoiesis, e.g. β-thalassemia, are associated with increased Epo and ROS concentrations implicated in the expansion of and damage to erythroid precursors, respectively. Treating erythroblasts with low dose ROS scavenger promotes enucleation, but high dose ROS scavenger leads to cell death [Zhao Exp Hematol 2016], suggesting that an optimal ROS concentration is integral to effective erythropoiesis. We and others have shown that ROS is increased in β-thalassemic erythroid precursors, but despite increased ROS, erythroid precursor apoptosis is not increased. We hypothesize that compensatory mechanisms prevent the ill-effects of increased ROS on erythroid precursors. Our prior experiments demonstrate disordered erythropoiesis in β-thalassemic (th1/th1) mice, restored in transferrin-treated th1/th1 mice [Liu Blood 2013], despite which, ROS remained increased in erythroid precursor from transferrin-treated th1/th1 mice. To identify mechanisms responsible for transferrin’s effect, we performed RNA seq analysis of erythroblasts from wild type (WT), th1/th1, and transferrin-treated th1/th1 mice. We identified increased pleckstrin-2 (plek2) in th1/th1 relative to WT mice, normalized in transferrin-treated th1/th1 mice. We hypothesize that plek2 activation counteracts the ill effects of ROS and promotes enucleation in β-thalassemia. Another currently funded collaborative R01 is aimed at exploring the role of plek2 in erythroid differentiation and enucleation in β-thalassemia (R01 NIDDK; PI: Ji).

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One main direction of the laboratory is to study the pathophysiology of iron overload and its regulation of / by erythropoiesis in iron loading anemias (e.g. β-thalassemia) using several mouse models (th1/th1 and th3/+). We hypothesized that transferrin, the main iron delivery molecule, provides an important compensatory mechanism in diseases of concurrent anemia and iron overload, demonstrating that exogenous transferrin ameliorates all erythroid- and iron-related pathology in th1/th1 mice [Li Nat Med 2010; Liu Blood 2013]. Evaluating hepcidin as a therapeutic tool [Gardenghi JCI 2010; Casu Blood 2016], its regulation in th3/+ mice in general [Parrow Blood 2012], and after treatment with transferrin [Chen haematol 2016], have further shed light on the mechanisms involved in exogenous transferrin’s effect on erythroid regulation of iron metabolism. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in β-thalassemic mice and that the beneficial effect of exogenous transferrin is mediated via decreased TfR1 expression. We previously demonstrated that apoTf-treated th1/th1 mice exhibit more iron restricted erythropoiesis, decreased transferrin saturation [Li Nat Med 2010], and less liver iron deposition [Chen haematol 2016]. Similar findings were evident in th3/+ mice [Gelderman haematol 2015]. Soluble TfR1, increased in β-thalassemic humans [Origa haematol 2007] and mice [Richardson Biochim Biophys Acta 1997], is decreased after treatment with apoTf in th1/th1 mice [Liu Blood 2013]. The current R01 funded work is aimed at exploring how TfR1 is involved in regulation of erythroid differentiation and enucleation in β-thalassemia (R01 NIDDK; PI: Ginzburg).