Loss of the Rho GTPase Activating Protein p190-B Enhances Hematopoietic Stem Cell Engraftment Potential.

Abstract

Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow (BM), self-renewal, proliferation and differentiation to mature blood cells. However, the molecular regulation of HSC engraftment is still poorly defined. Small Rho GTPases are critical regulator of cell migration, proliferation and differentiation in multiple cell types. p190-B GTPase Activating Protein (GAP) which regulates RhoA activity has been implicated in IGF-1 signaling via cAmp-response element binding proteins (CREB) activity regulating cell size during fetal development (Sordella, Dev Cell, 2002). Since IGF-1 and CREB play important roles in hematopoiesis, we hypothesize that p190-B is critical for HSC functions. Mice lacking p190-B die before birth. p190-B−/− embryos at 14.5 day post coitum exhibited fetal liver (FL) hypocellularity associated with profound anemia. Surprisingly, p190-B−/− FL showed normal frequency of myeloid colonies and a two-fold increase in day 28 and 35 CAFC frequency compared to WT. Overall, the number of progenitors per p190-B−/− liver was decreased while the number of HSC remained unchanged compared to WT FL, suggesting maintenance of the HSC pool in p190-B−/− FL despite the anemia. The repopulating capacity of p190-B−/− FL was assessed by serial competitive repopulation assay. Short-term repopulating ability of p190-B−/− FL was modestly increased compared to WT as assessed by peripheral blood (PB) chimerism 3 months post-transplant (58.3 ± 3.5% vs 45.5±5.4%, p<0.05, respectively). Remarkably, in secondary and tertiary recipients, p190-B−/− FL exhibited dramatically enhanced engraftment compared to WT (65.9±6.4% vs 29.3±6%, p<0.01; 21.7±7.9% vs 1.5±0.5%, p<0.05, respectively). The contribution to T cell, B cell and myeloid cell reconstitution was similar between the genotypes. BM analysis of secondary recipients revealed loss of the HSC pool in WT engrafted animals, while HSC frequency was near normal in p190-B−/− engrafted mice (LinnegScaposKitpos frequency: 0.034±0.019% vs 0.219±0.110%, p<0.05, respectively). Importantly, this enhanced long term engraftment was due to a difference in the functional capacity of p190-B−/− HSC compared to WT HSC since highly enriched p190-B−/− HSC (LSKMac1low) demonstrated similar enhanced engraftment in the BM 4 months post transplant. p190-B-mediated enhanced HSC engraftment was associated with normal proliferation and differentiation of p190-B−/− progenitors. Interestingly, a pool of quiescent HSC was maintained in serially transplanted p190-B−/− animals, since Pyronin Yneg LSK cells were still present in the BM of p190-B−/− engrafted mice while this population disappeared in WT controls. p190-B−/− FL cells also exhibited increased colony-forming unit homing to BM compared to WT controls. Together, these results suggest that loss of p190-B is associated with HSC quiescence and more efficient homing of HSCs that may contribute to long term regeneration of the hematopoietic stem cell pool after transplantation.