RASA3 Is Involved in Cell Cycle Progression, Hemoglobinization and Generation of Reactive Oxygen Species during Mammalian Erythropoiesis

RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. Defective RASA3 in zebrafish and mice results in severe anemia and thrombocytopenia. Indeed, in the mouse model scat (severe combined anemia and thrombocytopenia), a G125V mutation in Rasa3 leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Previous studies showed that this mutation in Rasa3 causes severely delayed erythroid differentiation at the polychromatophilic stage and decreased hemoglobinization due, at least in part, to mislocalization of RASA3 to the cytosol and resultant increased Ras activity. Here, we provide evidence that RASA3 plays a pivotal role in cell cycle progression and maintenance of reactive oxygen species (ROS) levels in erythropoiesis during crisis episodes. First, we analyzed the cell cycle progression in scat erythroblasts during crisis episodes. Using propidium iodide and flow cytometry, we found a significant increase in the G0/G1 phase (46.8% ± 3.1% in crisis vs 34.8% ± 2.5 in controls, p<0.001) while S phase was decreased (40.2% ± 2.9% vs. 49.6% ± 3.3% p<0.01) and G2 was not affected in scat proerythroblasts.  These data suggest that Rasa3 is involved in the G1 checkpoint. In addition, we observed an increase in the ROS levels in scat throughout differentiation including proerythroblasts (1.03% ± 0.3% vs. 0.25% ± 0.1% in controls, p < 0.01), EryA (1.5% ± 0.21% vs. 0.25% ± 0.05%, p < 0.01), EryB (2.6% ± 1.41% vs. 0.25% ± 0.01%, p < 0.01), EryC (2.0% ± 0.45% vs. 0.25% ± 0.02%, p < 0.01) and mature red cells (1.15% ± 0.1% vs. 0.9% ± 0.03%, p < 0.05); the highest levels of ROS were observed in the basophilic-polychromatic erythroblast (i.e. EryB) containing populations. In addition, we consistently found increased levels of ROS in both reticulocytes and red blood cells from scat peripheral blood. Therefore, the cell cycle arrest and increased ROS likely contribute to the erythropoietic defect associated with scat. No mutations involving RASA3 have been involved in human bone marrow failure syndromes yet. However, in more than 30% of the cases, the etiology remains unexplained. To understand the putative role of RASA3 in the pathophysiology of hematopoiesis, and based on our data obtained in the mouse and the zebrafish, we investigated the role of RASA3 in human erythropoiesis using si and shRNA knockdowns in cord blood-derived CD34+ cells.  Knockdown efficiencies were evaluated by western blot, and revealed that RASA3 protein levels were reduced by greater than 50% using two different shRNA constructs and siRNA.  Cells were differentiated using an adapted 3-phase liquid culture system that fully recapitulates erythropoiesis. Similar to the scat mouse, we observed delayed differentiation by flow cytometry using glycophorin A, band3 and α4-integrin as markers of terminal differentiation with 62.0% of RASA3 knockdown erythroblasts α4-integrin^hi (compared to 27.5% in controls) at day 16. In addition, the nucleus was substantially less pyknotic and the nucleocytoplasmic ratio remained elevated during differentiation, as compared to control cells. Indeed, 64 ± 7% of the cells had an uncondensed nucleus in the RASA3 knockdown samples, compared to 30 ± 3% in the scramble controls. Moreover as in scat, hemoglobinization was defective; qRT-PCR analyses revealed a 25% reduction in γ-globin and a 20% reduction in β-globin expression. These findings strengthen and emphasize the notion that RASA3 has a conserved function during vertebrate terminal erythropoiesis. Experiments investigating cell cycle progression, apoptosis and ROS production in human CD34+ cells are underway. Together, our studies demonstrate that RASA3 plays and important role in hemoglobinization, cell cycle progression and cell survival during terminal erythroid differentiation.