Rac1 and Rac2 Rho GTPases Distinctly Regulate Stem Cell Engraftment and Mobilization and Are Novel Targets for Pharmacologically-Induced Progenitor Mobilization.

Rac members of the Ras-related Rho GTPase family regulate mammalian cell cytoskeleton, survival and proliferation. We have recently implicated Rac1 in short-term hematopoietic stem/progenitor (HSC/P) cell engraftment and Rac2 in HSC/P mobilization (Gu Y et al., Science 2003). Indeed, Rac proteins are activated via β₁-integrins, CXCR4 and c-kit, all receptors implicated in homing and mobilization. Recent data examining the function of CXCR4 have been interpreted to show that mobilization and engraftment are mirror image processes. Using both a genetic and a pharmacological approach, we examined the role of Rac proteins in mobilization, engraftment and steady-state hematopoiesis. Here we demonstrate that whereas Rac1−/− HSC/P fail to engraft after transplantation, deletion of Rac1 by Cre-mediated deletion of floxed Rac1 after engraftment does not significantly affect either blood formation or HSC/P mobilization. Rac1−/−;Rac2−/− HSC/P dramatically fail to sustain steady-state hematopoiesis (over 95% reduction at 6 months) leading to a replacement of hematopoiesis by cells expressing Rac1. By infusing 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester labeled Lin-/c-kit+ BM cells into the blood followed by in situ fixative perfusion after 16 hours, we observed that the spatial distribution of transplanted marrow cells in the endosteal space (defined as less than 15 cells away from either endosteum in μm-longitudinal sections) was defective in Rac1−/− cells 4 (25%) versus wild-type (39%), suggesting defective retention of Rac1−/− HSC in the “stem cell niche” after engraftment. Rac1−/−;Rac2−/− lin-/c-kit+ cells showed a more severe defect in spatial localization to the endosteum (19% vs 39% in wild-type). In vitro, Rac1−/− HSC/P also showed a severely decreased cobblestone area formation ability (>95% reduction in CAFC frequency) but had normal transendothelial migration. In contrast, Rac2−/− HSC/P demonstrated normal short-term engraftment and only mild defects in these assays. Induction of combined Rac1 and Rac2 deficiency induces a striking mobilization of progenitor cells (Gu Y et al., Science 2003) while Rac1 re-expression by retrovirus-mediated gene transfer into these mobilized HSC is sufficient to effect engraftment of HSC/P into the BM (9-fold increase in engraftment ability in a competitive repopulation assay). Altogether these data suggest distinct roles for Rac1 versus Rac2 in retention of HSC/P in the BM endosteal space implying that engraftment and mobilization are not mirror image processes. To further exploit the identification of Rac as a regulator of HSC retention in BM, we employed a newly identified compound, NSC23776, specifically designed to block the interaction of Rac proteins with activating GTPase exchange factors (GEFs). When injected into the poorly mobilizing C57Bl/6 mouse strain, NSC23776 (2.5 mg/Kg i.p.) induced a 2-fold increase in circulating progenitors at 3–6 h after injection and additional trafficking (5-fold increase over wild-type mice) of these cells in Rac2−/− mice which show increased mobilization at baseline. The mobilization induced by a Rac inhibitory compound demonstrates that Rac is a novel target to induce mobilization of HSC/P.