Rho Gtpase Cdc42 Is Essential for Endothelial Niche Maintenance in the Bone Marrow and Hematopoiesis

Abstract 508

Adult hematopoietic stem/progenitor cells (HSPCs) are maintained in the unique microenvironment of bone marrow (BM) niche to sustain life-long blood production of mammals. Several types of cells in the BM, including endothelial cells (ECs), have been shown to contribute to the niche components in regulating the homeostasis of HSPCs as well as leukemia stem cells in the BM. However, because of the current technical limitations in studying BM ECs in mice, particularly the lack of an EC-specific inducible gene targeting method in adult mice and controversies surrounding the suitable murine EC culture conditions in vitro, the signaling pathways that regulate the putative BMEC niche remain poorly understood. Here we have utilized several complementary mouse models to achieve an inducible, BMEC-specific deletion of Cdc42 to unveil an essential role of this Rho family GTPase in regulating BMEC morphogenesis and proliferation, and in maintaining hematopoietic homeostasis of adult mice. First, by using the interferon-inducible Mx1-Cre;Cdc42lox/lox and the tamoxifen inducible Rosa26-CreER;Cdc42lox/lox mice engrafted with syngeneic WT HSPCs, we found that both conditions of inducible deletion of Cdc42 in the BM microenvironment, including BMECs, caused sinusoidal dilation, a drastic decline of endothelial capillary density, and a loss of endothelial progenitor activity and mature endothelial cells, in the bone marrow. Significantly, the BMEC phenotypes of these non-specific Cre-driver genetic models could be recapitulated by using a mouse model of Tie2-CreER;Cdc42lox/lox that allows inducible endothelial lineage-specific deletion of Cdc42 in the BM in response to tamoxifen, which we have shown rigorously by lineage tracing to be a suitable model for BMEC characterizations (Feng et al., ASH abstract, 2012). Second, we have established an in vitro culture system that expands and maintains primary BMECs in culture, allowing a direct assessment of the proliferation, survival, adhesion, migration, tube formation, as well as capillary assembly in 3D, activities of the BMECs. Deletion of Cdc42 by in vivo tamoxifen induction or in vitro Cre transduction of the BM cells of Tie2-CreER; Cdc42lox/lox genotype resulted in a complete loss of angiogenesis activity of BMECs that was associated with a drastic reduction of adhesion, tube-formation, and 3D capillary assembly capabilities of BMECs. In addition, endothelial cell proliferation was also compromised, as seen by a decrease of CyclinD1 expression and a diminished BrdU incorporation. These genetic results were phenocopied by findings using a Cdc42 specific inhibitor, CASIN, as CASIN treatment inhibited BMEC adhesion, diminished BM endothelial colony-forming activity, and suppressed BMEC tube-formation. A rescue experiment using retroviral expression of a panel of effector-coupling defective Cdc42 mutants and/or constitutively active PAK or AKT mutant in the Cdc42 knockout BMECs identified AKT and p21 PAK as the major downstream effectors of Cdc42 in BMEC maintenance. Third, using the three inducible mouse models, we found that Cdc42 in BMECs and microenvironment is required for maintaining hematopoietic homeostasis. Deletion of Cdc42 in BMECs caused a mobilization of HSPCs to the peripheral organs and a subsequent reduction of wild-type HSPCs in the BM, as assayed by genotypic FACS analysis, colony-forming activity and competitive transplantation of BM cells from endothelial Cdc42-null or control mice. Taken together, our results demonstrate that the Rho GTPase Cdc42 in BMECs mediates critical signaling cascade through PAK and Akt in maintaining the BMEC angiogenesis and morphogenesis. Cdc42 in BMECs could serve as a potential therapeutic target for bone marrow diseases including leukemia and lymphoma.