RASA3 Deficiency Contributes to Anemia By Multiple Mechanisms

RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a G125V mutation in Rasa3 that leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Our previous studies demonstrated that this mutation in Rasa3 causes defects in several aspects of erythropoiesis, including a significant delay of erythroid differentiation at the polychromatophilic stage, decreased hemoglobinization, defects in cell cycle progression past the G1 checkpoint, and increased reactive oxygen species (ROS) during terminal erythroid differentiation as well as in scat peripheral blood reticulocytes and red blood cells. We previously reported that the mislocalization of mutated RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the erythropoietic defect in scat, and the observed cell cycle arrest and increased ROS likely also contribute to this unique disease phenotype. Our current efforts are focused on further elucidation of the mechanism and specific disruptions in Ras signaling that lead to anemia, membrane fragmentation, and the cyclic phenotype in scat. Interestingly, we report here that apoptosis is not increased during scat crisis, and that mitochondria, a potential source of ROS, are normally eliminated at the reticulocyte stage.

The dramatic nature of remission, with complete normalization of all hematologic parameters, led us to hypothesize that a secreted factor may be mediating the cyclic phenotype of scat. Differences in the cytokine profile of the serum of scat mice compared to wild type suggest that, indeed, one or several secreted factor(s) may be influencing the occurrence of bone marrow failure. Levels of galectin-1, a known mediator of cell-cell interactions and intracellular signaling in the hematopoietic niche, are consistently decreased in scat serum according to a multispot anti-cytokine antibody array (23,326.5 ± 21,439.7 integrated density in scat vs. 31,019.6± 20,110.7 in controls, p<0.05).Studies exploring the influence of the galectin family on erythropoiesis and Ras signaling in the context of scat are underway.

Strengthening the notion that RASA3 has a critical conserved role in vertebrate terminal erythropoiesis, the characteristics of bone marrow failure seen in scat have been reproduced in human CD34+ cells using siRNA and shRNA knockdowns of Rasa3 . Similar to the changes seen in scat, cells with decreased RASA3 demonstrated delayed terminal erythroid differentiation and defective hemoglobinization. Finally, analysis of Ras expression and functional pull-down studies in human CD34+ cells revealed that, while K-Ras is the major active isoform expressed during terminal erythroid differentiation, H-Ras is also active during human erythropoiesis. Future studies with CD34+ Rasa3 knockdown cells will explore the influence of RASA3 on human K- and H-Ras signaling in erythropoiesis.

Taken together, our studies further characterize the vital role of RASA3 in hemoglobinization, cell cycle progression, and cell survival during terminal erythroid differentiation, as well as identify novel targets for investigation of unknown mechanisms (e.g., dysregulated cytokine secretion) of bone marrow failure syndromes.