The hematopoietic-specific rho gtpase, RAC2, is essential for normal adhesion and controlled movement of hematopoietic progenitor cells In vitroAnd In vivo

The mechanism of hematopoietic progenitor cell (HPC) migration and mobilization from bone marrow (BM) to peripheral blood (PB) has not yet been clearly defined. Since we have previously reported that absence of the Rho GTPase Rac2 results in defective movement and adhesion of mast cells and neutrophils, we speculated that the Rac2 protein might play a pivotal role in this process. Since previous reports have implicated the integrin VLA4 in HPC mobilization, we measured adhesion of purified HPC (mature cell lineage antigen negative (Lin−) c-kit+Sa-1+ cells isolated from Rac2−/− and wild type (WT) mice) to the VLA4 binding site, CS-1, on the recombinant HPC to this peptide was observed (21±5% in Rac2−/− cells, vs 61±8% in WT cells, P<0.001). In vitroanalysis of directed movement (modified Boyden chambers) by these cells towards stromal cell-derived factor-1 (SDF-1) demonstrated that, in contrast to the response of differentiated hematopoietic cells, migration of Rac2−/− HPC was increased 4-fold, compared with WT cells (12% versus 3% of cells migrated, respectively, P < 0.01). Increased movement was confirmed by time-lapsed video microscopy. Scanning electron microscopy showed that filopodia generated by these cells following stimulation with SDF-1 were much longer in Rac2−/− HPC compared with WT cells. In addition, Rac2−/− HPC was found to have increased f-actin content relative to WT. HPC movement in vivowas investigated by analysis of mobilization in Rac2−/− versus WT mice. Injection of 250μg of G-CSF/kg/day over 5 days resulted in mobilization of 2.8-fold more progenitors into PB of Rac2−/− mice compared with WT. Simultaneous injection of anti-VLA4 antibody with G-CSF into Rac2−/− mice resulted in significantly reduced augmentation of mobilization, compared with WT mice (in vitrocolony forming cells in PB=50% of WT; day 8 CFU-S in PB=69% of WT), consistent with defective VLA4-mediated function in Rac2−/− HPC. These data demonstrate that Rac2 is essential for normal adhesion and controlled movement of HPC, both in vivoand in vitro, and implicate this GTPase downstream of the integrin VLA-4. The data suggest that lack of Rac 2 in HPC may be associated with increased activity of other GTPases, possibly Cdc42.