A Critical Role of the Rho Family GTPase Cdc42 in Hematopoietic Stem Cell Mobilization, Homing, Engraftment and Differentiation.

Abstract

The Rho family GTPase Cdc42 has emerged as a key signaling molecule with unique roles in multiple hematopoietic cell lineages. To investigate the physiologic role of Cdc42 in hematopoietic stem cells/progenitors (HSC/Ps), we bred conditional cdc42flox/floxmice with Mx-Cre mice that allowed interferon-inducible cdc42 deletion in hematopoietic cells in vivo following polyI:C induction. Loss of Cdc42 expression in the bone marrow caused significant leukocytosis with neutrophilia in peripheral blood (PB) (Table). Concurrently, deletion of cdc42 in the bone marrow led to a massive efflux of HSC/Ps from bone marrow to PB, liver and spleen (Table) and increased proliferative activity of HSC/Ps. Consequently, flow analysis further revealed that the number of Lin-Sca-1+c-Kit+ (LSK) HSCs in BM, PB, spleen and liver increased significantly following cdc42 deletion (Table). Transplantation of the cdc42−/− bone marrow cells into NOD/SCID recipient mice or competitive transplantation into BoyJ recipient mice showed that cdc42 deficiency in the bone marrow caused the impaired engraftment (Table) and the failure of hematopoiesis after a transient increase of cell cycle rate of HSCs. The engraftment defect is associated with a homing deficiency (Table). The cdc42−/− (Lin−c-Kit+) HSC/Ps showed defects in cortical F-actin assembly, adhesion to fibronectin and directional migration in response to SDF-1α (Table). These data suggest that defective actin structure and adhesion of the cdc42−/− HSC/Ps may contribute to the loss of retention of the cells in the BM niche and to the mobilization phenotype. Moreover, deletion of cdc42 resulted in significantly reduced thymus cellularity, T cell, B cell population, and decreased Ter119+ cell number associated with markedly decreased CFU-E activity of the bone marrow cells (Table). Two additional complimentary approaches, BM-reconstitution by cdc42−/− HSCs and reciprocal (rescue) transplantation by WT HSCs, further demonstrated that the Cdc42 loss-of-function phenotypes are hematopoietic cell intrinsic. Taken together these results implicate Cdc42 as a critical regulator of HSC mobilization, homing and engraftment, and indicate that Cdc42 is involved in erythropoiesis and in T- and B-cell differentiation and homeostasis.WTCdc42−/−White blood cell count, × 109 /L11.0±2.540.8±3.9†Neutrophil count, × 109 /L4.6±0.834.1±4.7†CFU-C per 105 liver cells0.13±0.12169±132*CFU-C per 104 spleen cells1.36±1.0568.4±55.6**CFU-C per 100μl PB3.67±258.8±47.3**LSK HSCs in BM0.52%±0.22%0.94%±0.45%*LSK HSCs in PB0.13%±0.08%0.31%±0.15%*LSK HSCs in spleen0.37%±0.20%0.63%±0.23%*LSK HSCs in liver0.23%±0.10%0.70%±0.18%*Engraftment in NOD/SCID mice35.0%±4.9%23.0%±7.1%*Competitive transplantation61.5%±6.7%22.2%±2.9%**CFU-C Homing to BM (% injected)0.85%± 0.11%0.28%± 0.12%**Adhesion to fibronectin (%plating)50.8%±2.5%28.8%±4.0%**Migration to SDF (%input)26.2%±5.3%5.4%±0.9%†CD3+ T cells in BM5.4%±0.22%2.7%±0.45%**B220+ B cells in BM8.4%±4.6%1.5%±2.0%†Ter119+ cells in BM15.2%±9.3%6.0%±4.2%*CFU-E per 105 BM cells87.8±9.725.6±4.2†*p<0.05**p<0.005†p<0.001