Vesicular formation regulated by ERK/MAPK pathway mediates human erythroblast enucleation

Abstract

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.

Stage-specific dual function: EZH2 regulates human erythropoiesis by eliciting histone and non-histone methylation

“…Volcano and heat map analysis showed that the number of DEGs were approximately 419, with approximately 166 and 253 genes upregulated and downregulated in EZH2-knockdown cells, respectively (Figure 6B and C). Downregulated genes were enriched with GO terms involved in various biological processes reported to be tightly associated with nuclear condensation and enucleation, which occur specifically during the terminal stage of erythropoiesis [30][31][32] , including chromosome segregation, organelle fission and spindle organization. GSEA analysis revealed that the AURKB gene, a subunit of the chromosome guest protein complex, which ensures accurate chromosome segregation and cell division, was present in most of these key pathways (Figure 6D and E).…”

“…In this study, we are surprised to find that EZH2 regulates human erythropoiesis in a stage-specific dual function manner, by regulating early erythroid development via catalyzing H3K27me3 and modulating terminal stage development by eliciting non-histone methylation. It is important to note that terminal erythroid differentiation is a complex and highly regulated process that includes decreased nuclear size, chromatin condensation, and culminates in enucleation 31,32 .…”

“…It is important to note that terminal erythroid differentiation is a complex and highly regulated process that includes decreased nuclear size, chromatin condensation, and culminates in enucleation. 31 , 32 Previous studies have shown that some nuclear proteins, such as histones, are exported from the erythroid precursor nucleus into the cytoplasm and ultimately degraded during terminal erythroid development. 35 Our results demonstrated that H3K27me3 was exported into the cytoplasm of normal erythroblasts during maturation, whereas EZH2 was constantly localized in the nucleus during normal erythropoiesis.…”

Molecular regulatory mechanisms of erythropoiesis and related diseases

“…Previous research has demonstrated that vacuolar protein‐1 induces vesicle formation and increases the proportion of preadult erythrocytes with denuclei. When lattice, a regulator of endocytosis and vesicular transport, is inhibited in late juvenile erythrocytes, nucleus detachment is prevented, but red lineage cell proliferation and differentiation are unaffected 14 . Although vesicles can influence the detachment of nuclei, their precise mechanism of action remains unknown.…”

“…rocytes, nucleus detachment is prevented, but red lineage cell proliferation and differentiation are unaffected. 14 Although vesicles can influence the detachment of nuclei, their precise mechanism of action remains unknown.…”