Abstract
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.
Highlights
Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation
Erythroblast reactive oxygen species (ROS) are increased in βthalassemic mice[13,28] and prior reports suggest that ineffective erythropoiesis in β-thalassemia is a consequence of increased ROS11, we demonstrate that reversal of ineffective erythropoiesis in apoTf-treated β-thalassemic mice occurs as a consequence of decreased ROS in pro-erythroblasts (Pro-E) stage, despite persistently increased ROS in later erythroblasts (Fig. 1a)
The fraction of enucleated erythroblasts is unchanged in thal plek2+/− relative to thal mice (Fig. 2i). These results demonstrate that the fraction of thal plek2+/− that survive to analysis are the least pathologically affected and that erythroblast apoptosis is increased despite increased Epo in thal plek2+/− mice, together revealing that ineffective erythropoiesis is mitigated by upregulation of plek[2] in β-thalassemic erythropoiesis
Summary
Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. 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. A complete mechanistic understanding of how apoTf improves ineffective erythropoiesis is unavailable, evaluating apoTf-treated βthalassemic mice provides an opportunity to explore mechanisms underlying ineffective erythropoiesis and its reversal to better understand regulatory nodes in ineffective erythropoiesis. In this current work, we identify pleckstrin-2 (plek2) as an essential response element in β-thalassemic mice and use plek[2] knockout mice to generate plek[2] loss in β-thalassemic mice. Several previously published manuscripts provide data on the functional importance of plek[2] in erythropoiesis
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