RASA3 Plays a Critical, Conserved Role in Erythroid Differentiation

Abstract 3186

Identification of new key players in erythropoiesis can lead to a better understanding of the etiology of anemia of unknown origin. Mouse models have significantly contributed to our understanding of normal erythropoiesis and the pathogenesis of erythroid disorders. Recently, we identified in the scat (severe combined anemia and thrombocytopenia) mouse model a missense mutation (G125V) in the Rasa3 gene, encoding a Ras GTPase activating protein (GAP). Homozygous scat mice present a cyclic phenotype with alternating episodes of crisis and remission. Crisis episodes are characterized by severe anemia and thrombocytopenia while, remarkably, the phenotype reverts to normal in the remission phase. Remissions are transient, however, and 94% of scat/scat mice die by P30 during a second crisis episode. We recently demonstrated a mechanism contributing to crisis episodes. The G125V mutation in Rasa3 is a loss of function mutation causing the protein to be mislocalized to the cytosol in scat reticulocytes. This results in loss of GAP activity and, as a consequence, increased levels of active GTP-bound Ras and a severe block in erythroid differentiation. Morpholino knockdowns of rasa3 in zebrafish result in profound anemia, confirming a conserved and non-redundant role for Rasa3 in vertebrate erythropoiesis. Here, we report that the cell cycle is affected in scat erythroid progenitors and extend studies to human primary erythroid cells. Using propidium iodine 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, × ± SD 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. To determine if RASA3 function is conserved in human erythropoiesis, we isolated CD34+ cells from human cord blood and cultured them using the recently described three-phase culture system that recapitulates human erythropoiesis over a period of approximately 21 days. By western blotting, we found that RASA3 protein levels increase during the early stages of differentiation, peaking at Day 11, before decreasing continuously as terminal erythroid differentiation proceeds. We also used siRNA to transiently knock-down RASA3 during differentiation. Cells were transfected at D8 or D11, and effects of RASA3 silencing were evaluated after a 72 hours incubation period, at D11 or D14. The transfection efficiency was close to 90% and knock-down of RASA3 was verified at the protein level. While we did not see any apparent changes in terms of proliferation, as judged by cell counts and total protein content, the siRNA treated cells presented with a delay in differentiation as compared to control (scramble siRNA). Specifically, morphologic analyses of the cells by Giemsa and benzidine staining demonstrated that nuclear condensation and hemoglobinization were clearly affected, especially at D14. In the RASA3 siRNA transfected cells, the nucleus was substantially less pyknotic and the nucleocytoplasmic ratio did not decrease during differentiation, as compared to cells before transfection or transfected with scramble siRNA, and benzidine staining revealed a significant decrease in the extent of hemoglobinization. A lack of nuclear condensation and defects in hemoglobinization are consistent with a delay in differentiation, and extend the paradigm observed in the scat mouse model to human erythropoiesis.