Role of Activated Pleckstrin-2 and Down-Stream Effects on Ineffective Erythropoiesis in β-Thalassemic Mice

Erythropoiesis involves stem cell differentiation to mature red blood cells (RBCs). Erythropoietin (Epo) is essential for erythroipoiesis, and 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.i 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,i 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,ii 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. Using confocal microscopy, we demonstrate that 1) plek2 co-localizes with actin on the cell membrane after the pro-erythroblast stage but is in the nucleus throughout terminal erythropoiesis in WT mice; 2) membrane-associated plek2 is present earlier, in pro-erythroblasts, and remains membrane-associated until orthochromatophilic stage in th1/th1 mice; and 3) plek2 localization is normalized in transferrin-treated th1/th1 mice. Because plek2 activation leads to its association with the cell membrane and plek2 activation is increased in th1/th1 erythroblasts, we set out to explore the role of plek2 activation on ineffective erythropoiesis in transferrin-treated th1/th1 mice. Prior publications propose that plek2 interacts with and results in the phosphorylation of cofilin, preventing cofilin's translocation to the mitochondria as part of the apoptosis pathway in response to increased ROS.iii We demonstrate decreased in mitochondria cofilin localization and increased cellular p-cofilin in th1/th1 erythroblasts, normalized after transferrin treatment. These data suggest that despite an increase in ROS, plek2 and its induction of p-cofilin inhibit apoptosis in β-thalassemic erythroid precursors. Furthermore, in light of a prior report of an anti-enucleation effect of plek2 in vitroiii and the known regulation of enucleation by RacGTPasesiv, we hypothesize that plek2 activation triggers RacGTPase and prevents enucleation in th1/th1 mice. We demonstrate that in addition to changes in erythroblast RacGTPase concentration, membrane co-localization between plek2 and RacGTPase is enhanced and occurs earlier in th1/th1 erythroid differentiation relative to WT, normalized after transferrin treatment. Lastly, cleavage of Rho-associated kinase, Rock1, associated with enucleation,v is also decreased in th1/th1 erythroblasts, enhanced after transferrin treatment. Taken together, we speculate that plek2 haplo-insufficiency benefits β-thalassemic mice by enabling apoptosis of ineffective erythroblasts, or result in worsening,

possibly lethal, phenotype in light of the direct or indirect (through effects on RacGTPase or Rock1) role of plek2 in enucleation. Presently, we are testing this hypothesis by generating plek2+/- and plek-/- β-thalassemic mice. In conclusion, we demonstrate the important compensatory role of plek2 in β-thalassemic erythropoiesis. i Zhao Exp Hematol 2016

ii Liu Blood 2013 iii Zhao haematol 2014

iv Ji Nat Cell Bio 2008

v Gabet Cell Death Diff 2011