Knock-Down of RasGRP3 Expression Impairs Migration Towards SDF-1 and Increases Cell Size in HEL Cells

Background Umbilical cord blood (UCB) transplants in patients with hematological and non-hematological malignancies are impaired by suboptimal bone marrow (BM) homing of hematopoietic stem progenitor cells (HSPCs). We have previously reported that Ras guanine nucleotide-releasing protein 3 (RasGRP3), a protein responsible for GDP/GTP exchange of Ras, regulates the transmigration ability of human UCB CD34+ cells. UCB CD34+ cells that migrated toward stromal derived factor-1 (SDF-1) in transwell assays, showed an elevated RasGRP3 and decreased erythropoietin receptor (EPOR) gene expression. In this study, we examine the role of RasGRP3 protein in the in vitro migration of a RasGRP3 and EPOR expressing human erythroleukemia cell line, HEL 92.1.7 (HEL). Methods HEL cells were transduced with a pLKO.1 GFP shRNA plasmid that is specific and efficient for gene knockdown. Cells were also transduced with a plasmid expressing a full length of untagged human RasGRP3, which overexpressed RasGRP3. Cells were sorted by a BD FACSAria II for their specific fluorophore marker and cultured in RPMI + IMDM + pen/strep + 5% fetal bovine serum. “Empty” vectors with just the fluorescent marker were used for controls. RasGRP3 levels were also measured using western blotting.The migration assay was set up using cell inserts with 12uM pores. For each cell type there was a control well with no chemoattractant and then the well with 125 ng/ml of SDF-1. 100,000 cells HEL cells were seeded into culture wells per condition. The plate was then left undisturbed for 24 hours in the 37°C incubator. After 24 hours, the inserts were removed and the wells in the plate were counted for cells using a hemocytometer. Results Overexpressing RasGRP3 in HEL cells resulted in an increase in RasGRP3 protein (75 kDa) expression and an increase in a 25kDa band that corresponds to a RasGRP3 degradation product as we have seen in the past and according to the antibody manufacturer Cell Signaling. Knocking down RasGRP3 in HEL cells resulted in the elimination of the 25kDa band (Figure-1). Knocking down RasGRP3 in HEL cells eliminated the migration potential toward SDF-1, p=0.0045 (Figure 2). However, no change in migration was seen in overexpressing RasGRP3. In ImageStream analysis, it was seen that RasGRP3 knockdown cells were larger than the control cells, while RasGRP3 overexpressed cells were smaller than the control cells (Figure 3). Conclusions RasGRP3 plays an important role in the migration of the EPOR expressing HEL cells toward SDF-1. We have shown that knocking out RasGRP3 using a shRNA lentiviral system eradicates migration in HEL cells. We can tentatively explain the decrease in migration by the larger cell size seen in knock-down cells, which might impair migration in a 3-dimentional environment. Next steps would be to find the mechanism of RasGRP3 effect on cell size, and to improve migration of HSPCs by targeting RasGRP3.