A Critical Role of Reactive Oxygen Species In the Generation of Megakaryocyte-Erythrocyte Progenitor Cells.

Abstract 1605

Reactive oxygen species (ROS) are small molecules containing oxygen with unpaired electron. ROS are always generated as the products of cellular metabolism, and cells have several antioxidant systems to avoid their harmful effect induced by their high chemical reactivity. While studies about biological effects of ROS have been mainly focused on the harmful aspects, a growing body of evidence suggests that ROS are critical mediators of several signaling pathways in cellular homeostasis. In hematopoiesis, for example, it was reported that the generation of intracellular ROS functions as the initiation signal in the development of Drosophila hematopoietic cells and that ROS control self-renewal of hematopoietic stem cells. However, little is known about the functions of ROS in regulating hematopoietic progenitors. In this study, we show a critical role of ROS in lineage decision of myeloid progenitor cells. Firstly, we measured the intracellular ROS level of hematopoietic cells from murine bone marrow by flow cytometry with H2-DCFDA (2’,7’-dichlorofluorescin diacetate) staining. It was found that intracellular ROS level of megakaryocyte-erythrocyte progenitor cells (MEP) was kept equal to or lower than that of lineage marker negative, Sca-1 positive, c-Kit positive (KSL) cells. On the other hand, that of granulocyte-monocyte progenitor cells (GMP) was significantly elevated. Additionally, mRNA expression of NADPH oxidase 2 (cytochrome b-245) and NOXA2 (neutrophil cytosolic factor 2), both of which are major components of the membrane-bound oxidase complex generating ROS in cells, was significantly suppressed in MEP and KSL cells, whereas it was up-regulated in GMP. Thus, intracellular ROS level of MEP is kept lowest in hematopoietic cells. Next, we investigated the effect of ROS on the differentiation of myeloid progenitors. In liquid culture assay, loading of ROS with low dose hydrogen peroxide inhibited the differentiation of progenitor cells into MEP, whereas removal of ROS with catalase accelerated the differentiation of those into MEP. Similarly, in the colony-forming assay with semisolid culture medium, we observed that loading of ROS inhibited the formation of megakaryocyte-erythrocyte colonies. To investigate the effect of intrinsic ROS on the colony-forming capacity, we sorted ROS-high and low common myeloid progenitor cells (CMP) individually and cultured them. It was shown that ROS-low CMP had high colony-forming capacity of megakaryocyte-erythrocyte, whereas ROS-high CMP had high colony-forming capacity of granulocyte-monocyte. There is inverse correlation between intracellular ROS level of CMP and colony-forming capacity of megakaryocyte-erythrocyte. In order to confirm that ROS can control the differentiation of CMP into MEP or GMP in vivo, we injected lipopolysaccharide (LPS), which can increase intracellular ROS level of bone marrow cells, into mice. We found that MEP were decreased in LPS-injected mice. Finally, we performed gene expression microarray analysis to compare gene expression profiles between ROS-low and high CMP. In terms of gene expression profiles, ROS-low CMP were similar to MEP and magakaryocyte whereas ROS-high CMP were similar to GMP. In conclusion, these findings suggest that intracellular ROS play a critical role in lineage decision of myeloid progenitor cells, especially in the generation of MEP. In several diseases with anemia or thrombocytopenia, such as myelodysplastic syndrome, Fanconi anemia and autoimmune diseases with chronic inflammation, it was reported that intracellular ROS level of hematopoietic cells was up-regulated. Thus, up-regulated intracellular ROS may be involved in their symptoms via differentiation disorder of MEP.