Genetic Evidence for a Role of Src Kinases in Mobilization, Homing and Engraftment of Hematopoietic Stem Cells: Deficiency of Src Kinases Results in Enhanced G-CSF Induced Mobilization.

Although it is clear that chemokines, cytokines and adhesion molecules play an essential role in regulating hematopoietic stem and progenitor cell (HSC/P) self-renewal, lineage commitment, apoptosis and mobilization, the intracellular signals that regulate these processes are poorly defined. Here, we demonstrate that the deficiency of hematopoietic specific Src family kinases (SFKs) in Lin- HSC/Ps results in reduced chemotaxis and adhesion via CXCR4 and β1 integrins, respectively (n=3, p<0.01). The reduction in SDF-1 induced chemotaxis was observed in spite of elevated CXCR4 expression on SFK−/ − HSC/Ps compared to wildtype controls (51% vs 69%, n=3, p<0.05). In vivo, the defect in CXCR4 and β1 signaling resulted in a significant increase in steady-state mobilization of SFK−/ − HSC/Ps in the spleen and peripheral blood (n=6, p<0.01). Remarkably, defective chemotaxis via CXCR4 and adhesion via β1 in SFK−/ − HSC/Ps was associated with a two and a twenty-fold increase in anti-α4β1 and G-CSF induced peripheral blood (PB) mobilization, respectively (n=16, p<0.001). Previous studies have shown that G-CSF induced mobilization is mediated in part by the release of neutrophil proteases in the bone marrow (BM), which cleave SDF-1 and VCAM-1 thereby disrupting CXCR4/SDF-1 and VCAM-1/α4β1 interactions. We hypothesized that the enhanced G-CSF induced mobilization due to SFK deficiency may partly be due to SFKs role in negatively regulating the expression of neutrophil proteases, thereby resulting in enhanced degradation of SDF-1 and VCAM-1. Consistent with our hypothesis, incubation of G-CSF treated BM fluid from SFK−/ − mice demonstrated a five-fold higher proteolytic activity towards SDF-1 and VCAM-1 compared to wildtype controls. In contrast, although anti-α4β1 induced mobilization of HSC/Ps also resulted in the degradation of SDF-1; no affect on the degradation of VCAM-1 was observed. These results suggest, that although, both G-CSF and anti-α4β1-treatment results in the mobilization of HSC/Ps, the affect of G-CSF on the degradation SDF-1 and VCAM-1 is more profound. Although the number of BM derived progenitors in response to various cytokines was significantly less in SFK−/ − mice compared to wildtype, SFK−/ − BM cells were hypersensitive to G-CSF. To assess the affect of an altered BM microenvironment on the long-term repopulating ability and multilineage engraftment of SFK−/ − stem cells, we performed a competitive repopulation assay. Interestingly, BM derived SFK−/ − progenitors homed significantly better than wildtype cells in a short-term homing assay. The increased homing of SFK−/ − BM cells was due to hyper induction of α4β1, α5β1 and CD43 on BM homed SFK−/ − cells compared to wildtype (n=6, p<0.002). Furthermore, the overall myeloid cell engraftment was also significantly enhanced in mice transplanted with SFK−/ − stem cells compared to wildtype controls in both primary and secondary recipients (n=32, p<0.005). In contrast, lymphoid cell chimerism, in particular B cell engraftment was significantly reduced in both primary and secondary recipients transplanted with SFK−/ − stem cells (n=32, p<0.005). Taken together, our results demonstrate that SFKs play an essential role in regulating multiple HSC/P cell functions, including homing, mobilization, and multi-lineage commitment. Thus, targeting SFKs may be of therapeutic importance for modulating both growth and actin cytoskeletal functions in HSC/Ps.