The Actin Polymerization Regulator WAVE2 Is Required for Early Bone Marrow Repopulation by Hematopoietic Stem Cells

Abstract

The Rho GTPase family members play essential roles in hematopoiesis. Of these, Rac1 is thought to be required for the appropriate spatial localization of hematopoietic stem and/or progenitor cells (HSPCs) within the bone marrow (BM), whereas Rac2 likely plays a role in BM retention of HSPCs. To elucidate the molecular mechanisms underlying Rac-mediated functions in hematopoietic stem cells (HSCs), we studied Wiskott-Aldrich syndrome protein family verprolin-homologous proteins (WAVEs), the specific effectors downstream of the Rac GTPases in actin polymerization. We here showed that CD34 2/low cKit 1 Sca-1 1 lineage 2 HSCs (CD34 2 KSL HSCs) express WAVE2 but neither WAVE1 nor WAVE3. Because WAVE2 knockout mice are embryonic-lethal, we utilized HSCs in which the expression of WAVE2 was reduced by small interfering RNA. We found that knockdown (KD) of WAVE2 in HSCs affected neither in vitro colony formation nor cell proliferation but did impair in vivo long-term reconstitution. Interestingly, WAVE2 KD HSCs exhibited unaltered homing but showed poor BM repopulation detected as early as day 5 after transplantation. The mechanistic studies on WAVE2 KD HSCs revealed modest but significant impairment in both cobblestone-like area-forming on stromal layers and actin polymerization upon integrin ligation by fibronectin. These results suggested that WAVE2-mediated actin polymerization, potentially downstream of Rac1, plays an important role in intramarrow mobilization and proliferation of HSCs, which are believed to be crucial steps for long-term marrow reconstitution after transplantation.

Vinculin Is Indispensable for Repopulation by Hematopoietic Stem Cells, Independent of Integrin Function

“…Transplantation of HSC populations has been shown to be sufficient for long term multilineage reconstitution, not only in experimental animal models but also in clinical patients with hematological malignancies. HSCs must undergo several steps to achieve engraftment after transplantation, including transendothelial migration into the BM (homing), settling in the BM niche (lodging and retention), and intra-BM proliferation and multilineage differentiation (repopulation) (30). The present study showed that HSCs lacking vinculin were unable to reconstitute the hematopoietic sys- tem efficiently and failed to maintain their self-renewal capacity on BM stromal cell layers.…”

“…Cobblestone-like Area Forming Assay-Cobblestone-like area forming assays were essentially performed as reported previously (30). C3H/10T1/2 cells were cultured in 96-well plates and then irradiated with a single dose of 50 grays.…”

Human CD34 + /KDR + Cells Are Generated From Circulating CD34 + Cells After Immobilization on Activated Platelets

“…First, adherent CD34 ϩ cells ( Figure 2D) exhibited dense areas of polymerized filamentous actin (F-actin) in the cortical cytosol, demonstrating the activated status of the cells, which is secondary to PI3K-dependent activation involving Rac1, protein kinase C-, extracellular signal-regulated kinase 1/2, and WAVE2. 11 In contrast, nonsheared CD34 ϩ cells show diffuse low-level staining of F-actin ( Figure 2C).…”

Megakaryocyte-matrix interaction within bone marrow: new roles for fibronectin and factor XIII-A

“…Interestingly, FN is a major component of bone marrow ECM and may modulate homing of hemopoietic progenitor cells 41 and the organization and composition of bone marrow ECMs and cell-matrix adhesion sites. 40 The present work suggests a new role for MKs as promoters of matrix deposition and remodeling within the bone marrow environment, with particular emphasis on the type I collagen-rich osteoblastic niche.…”