The Effect And Mechanism Study Of Altered Autophagy On Megakaryopoiesis

Autophagy is an effective strategy for cell development by recycling cytoplasmic constituents by degradation of bulk plasma, long-lived proteins and organells. Loss of autophagy in hematopoietic stem cells (HSCs) can lead to failure of megakaryopoiesis and enhanced autophagy has been implicated in various hematological disorders such as immune thrombocytopenia and myelodysplastic syndrome. Therefore, people began to focus on the study of the effect of autophagy on megakaryopoiesis and platelet formation recently. In order to explore the regulation of autophagy on megakaryopoiesis and thrombopoiesis, we used both autophagy inducer and inhibitor to regulate autophagy in vitro together with the megakaryocyte-and platelet-specific deletion of autophagy-related gene Atg7 mice model to see the alteration of megakaryocytes and platelets.ObjectiveExploring the possible role, specific targeted stage and mechanism of altered autophagy in megakaryocyte development.MethodsWe realized the alterations of autophagy in early or late megakaryopoiesis and cell-specific deficiency of autophagy using pharmacological approaches (induction by rapamycin or inhibition by bafilomycin Al) and megakaryocyte- and platelet-specific deletion of autophagy-related gene Atg7 mice model, respectively.1. In vitro study:we regulated autophagy in hematopoietic stem cells and mature megakaryocytes successfully using rapamycin and bafilomycin Al, which was confirmed by measuring LC3-II expression. Meanwhile, we examined some specific markers in early or late megakaryopiesis, such as the maturation of both nucleus and cytoplasma, expression of megakaryocytic markers, proplatelet formation and platelet release.2. In vivo study:we calculated the platelet number between Atg7flox/flox PF4-Cre and Atg7flox/flox mice, compared their megakaryocytes, and monitored their platelet recovery after low dose X-ray irradiation induced thrombocytopenia.3. Exploration of preliminary mechanisms:we used western blot and Q-PCR to examine the potential factors involved in the regulation of autophagy on megakaryopoiesis.ResultsWhen autophagy was induced by rapamycin or inhibited by bafilomycin A1 in fetal liver cells, we observed significant decreases in high ploidy megakaryocytes (Rap 54.8±0.6%, BafA 50.4±3.8%, Ctrl 71.1±2.0%, P?0.05), reductions in CD41 and CD61 dual-positive cells (Rap 1.0±0.1%, BafA 0.7±0.0%, Ctrl 5.2±0.1%, P?0.001), and reduced platelet release from megakaryocytes (Rap 0.4±0.3%, BafA 0.7±0.1%, Ctrl 8.5±0.5%, P<0.05), compared with the control cells with basal level of autophagy, which showed decreased ploidy and co-expression of CD41 and CD61 in megakaryocytes derived from mouse fetal liver cells treated with rapamycin or bafilomycin A1. Meanwhile, we found decreased expression of transcriptional factor GATA-1 (Rap 0.56±0.14, P?0.05; BafA 0.40±0.06, P?0.01), and cell cycle related proteins, such as CyclinDl (Rap 0.44±0.03, P?0.001; BafA 0.68±0.06, P?0.01), CyclinD2 (Rap 0.25±0.03, P?0.001; BafA 0.35±0.02, P?0.05) and P21 (Rap 0.04±0.02, P?0.05; BafA 0.04±0.02, P?0.05) in megakaryocytes derived from rapamycin- or bafilomycin A1-treated fetal liver cells compared with the control cells (GATA-1:Ctrl 1.15±0.13; CyclinD1:Ctrl 1.0±0.07; CyclinD2:Ctrl 0.49±0.03; P21:Ctrl 0.16±0.03). Megakaryocyte size was also reduced by rapamycin, but not by bafilomycin A1. Western blot analysis of revealed that treatment by rapamycin promoted the expression of phospho-MLC.However, when autophagy was altered in mature megakaryocytes, we observed no significant change in proplatelet formation, which was consistent with normal platelet counts, megakaryocyte numbers, ploidy and platelet recovery after low dose X-ray irradiation in Atg7flox/flox PF4-Cre mice with megakaryocyte-and platelet-specific deletion of autophagy-related gene Atg7.In all, our findings suggest that either induction or inhibition of autophagy in early stage of megakaryopoiesis suppresses megakaryopoiesis and thrombopoiesis and targeting autophagy may provide therapeutic potential for dysmegakaryopoiesis and thrombocytopenia.Conclusion1. Either induction or inhibition of autophagy in early stage of megakaryopoiesis suppresses megakaryopoiesis and thrombopoiesis, which is due to the regulation of transcriptional factor GATA-1, cell cycle related proteins CyclinD1, CyclinD2 and P21, and the downstream molecule of RhoA, phospho-MLC.2. Altered autophagy in mature megakaryocytes does not affect proplatelet formation, and loss of autophagy in megakaryocytic lineage does not influence meagakaryopoiesis and platelet recovery after low dose X-ray irradiation.