Enucleation is a key event in mammalian erythropoiesis responsible for generation of enucleated reticulocytes. While progress is being made in developing mechanistic understanding of enucleation, our understanding of mechanisms for enucleation is still incomplete. Mitogen-activated protein kinase (MAPK) pathway plays diverse roles in biological processes but its role in erythropoiesis is yet to be fully defined. Analysis of RNA-seq data revealed that MAPK pathway is significantly up regulated during human terminal erythroid differentiation. MAPK pathway consists of three major signaling cassettes, MEK/ERK, p38 and c-Jun N-terminal Kinases (JNK). In the present study, we show that amongst these three cassettes, only ERK was significantly up regulated in late stage human erythroblasts. The increased expression of ERK along with its increased phosphorylation suggests a potential role of ERK activation in enucleation. To explore this hypothesis, we treated sorted populations of human orthochromatic erythroblasts with MEK/ERK inhibitor U0126 and found that U0126 inhibited enucleation. In contrast, inhibitors of either p38 or JNK had no effect on enucleation. Mechanistically, U0126 selectively inhibited formation/accumulation of cytoplasmic vesicles and endocytosis of the transferrin receptor without affecting chromatin condensation, nuclear polarization and enucleosome formation. Treatment with vacuolin-1 that induces vacuole formation partially rescued the blockage of enucleation by U0126. Moreover, phosphoproteomic analysis revealed that inactivation of the ERK pathway led to down regulation of endocytic recycling pathway. Collectively, our findings uncovered a novel role of ERK activation in human erythroblast enucleation by modulating vesicle formation and have implications for understanding anemia associated with defective enucleation.
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