IntroductionThe majority of hematopoietic progenitor cells (HPCs) reside in the bone marrow surrounded by a complex, highly organized microenvironment. Under normal conditions, a small number of HPCs are released into the peripheral blood. Agents with distinct cellular targets and biologic activities can induce the mobilization of HPCs into blood, including hematopoietic growth factors, chemotherapeutic agents, and chemokines. 1,2 Recently, mobilized peripheral blood HPCs have become the principal cellular source for reconstitution of the hematopoietic system following myeloablative therapy. Currently, granulocyte colony-stimulating factor (G-CSF) is the most widely used agent to induce HPC mobilization due to its potency, predictability, and safety. 3 However, the mechanisms responsible for G-CSF-induced HPC mobilization have not been defined.We previously showed that G-CSF receptor (G-CSFR) expression on HPCs is not required for their mobilization by G-CSF, suggesting that G-CSF induces HPC mobilization indirectly through the generation of trans-acting signals. 4 The nature of the transacting signals that mediate G-CSF-induced HPC mobilization is unknown; however, accumulating evidence suggests that interaction of CXCL12 (stromal-derived factor 1 [SDF-1]) with its cognate receptor, CXCR4 (CXC motif, receptor 4), may play an important role in regulating G-CSF-induced HPC mobilization. CXCL12 is a CXC chemokine constitutively produced in the bone marrow by stromal cells. 5 Studies of CXCL12-or CXCR4-deficient mice have established that these genes are necessary for the normal migration of HPCs from the fetal liver to the bone marrow and in the efficient retention of myeloid precursors in the adult bone marrow. 6,7 Moreover, treatment with AMD-3100, a specific antagonist of CXCR4, induces rapid and robust HPC mobilization in both humans and mice. 8,9 Finally, we and others showed that CXCL12 protein expression in the bone marrow is significantly decreased following G-CSF treatment. [10][11][12] Collectively, these data suggest a model in which disruption of CXCL12/CXCR4 signaling is a key step in G-CSF-induced HPC mobilization.The mechanisms mediating the G-CSF-induced decrease in CXCL12 protein expression in the bone marrow have not been For personal use only. on May 12, 2018. by guest www.bloodjournal.org From defined. Previous reports suggested that neutrophil elastase (NE) and cathepsin G (CG) might regulate CXCL12 protein expression in the bone marrow through proteolytic cleavage of CXCL12. 10,11 However, mice genetically lacking NE and CG display normal G-CSF-induced HPC mobilization, and the expected decrease in bone marrow CXCL12 protein was observed. 13 Thus, the G-CSFinduced decrease in CXCL12 protein expression in the bone marrow does not require these proteases. It is possible that other proteases can compensate for the loss of NE and CG. Alternatively, nonproteolytic mechanisms may regulate CXCL12 expression in the bone marrow during G-CSF-induced HPC mobilization.In this study, we characterize G-CSF...

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.

The modeling and remodeling of bone requires activation and polarization of osteoclasts, achieved by reorganization of the cytoskeleton. Members of the Rho subfamily of small GTPases, including Cdc42, are known regulators of cytoskeletal components, but the role of these proteins in bone physiology and pathophysiology remains unclear. Here, we examined loss-of-function mice in which Cdc42 was selectively ablated in differentiated osteoclasts and gain-of-function animals wherein Cdc42Gap, a protein that inactivates the small GTPase, was deleted globally. Cdc42 loss-of-function mice were osteopetrotic and resistant to ovariectomy-induced bone loss, while gain-of-function animals were osteoporotic. Isolated Cdc42-deficient osteoclasts displayed suppressed bone resorption, while osteoclasts with increased Cdc42 activity had enhanced resorptive capacity. We further demonstrated that Cdc42 modulated M-CSF-stimulated cyclin D expression and phosphorylation of Rb and induced caspase 3 and Bim, thus contributing to osteoclast proliferation and apoptosis rates. Furthermore, Cdc42 was required for multiple M-CSF-and RANKL-induced osteoclastogenic signals including activation and expression of the differentiation factors MITF and NFATc1 and was a component of the Par3/Par6/atypical PKC polarization complex in osteoclasts. These data suggest that Cdc42 regulates osteoclast formation and function and may represent a promising therapeutic target for prevention of pathological bone loss.

Maintenance of bone mass and integrity requires a tight balance between resorption by osteoclasts and formation by osteoblasts. Exocytosis of functional proteins is a prerequisite for the activity of both cells. In the present study, we show that synaptotagmin VII, a calcium sensor protein that regulates exocytosis, is associated with lysosomes in osteoclasts and bone matrix protein-containing vesicles in osteoblasts. Absence of synaptotagmin VII inhibits cathepsin K secretion and formation of the ruffled border in osteoclasts and bone matrix protein deposition in osteoblasts, without affecting the differentiation of either cell. Reflecting these in vitro findings, synaptotagmin VII-deficient mice are osteopenic due to impaired bone resorption and formation. Therefore, synaptotagmin VII plays an important role in bone remodeling and homeostasis by modulating secretory pathways functionally important in osteoclasts and osteoblasts.

Estrogen-deficient osteoporosis may be an inflammatory disorder and we therefore asked if IL-17 participates in its pathogenesis. Deletion of the principal IL-17 receptor (IL-17RA) protects mice from ovariectomy (OVX)-induced bone loss. Further supporting a central role of IL-17 in its pathogenesis, OVX-induced osteoporosis is prevented by a blocking antibody targeting the cytokine. IL-17 promotes osteoclastogenesis by stimulating RANK ligand (RANKL) expression by osteoblastic cells, mediated by the IL-17RA SEFIR/TILL domain. Estrogen deprivation, however does not enhance IL-17RA mRNA expression by osteoblasts or in bone, but augments that of Act1, an IL17RA-interacting protein and signaling mediator. Similar to IL-17RA−/− mice, those lacking Act1 are protected from OVX-induced bone loss. Also mirroring IL-17RA-deficiency, absence of Act1 in osteoblasts, but not osteoclasts, impairs osteoclastogenesis via dampened RANKL expression. Transduction of WT Act1 into Act1−/− osteoblasts substantially rescues their osteoclastogenic capacity. The same construct, however, lacking its E3 ligase U-box or its SEFIR domain, which interacts with its counterpart in IL-17RA, fails to do so. Estrogen deprivation, therefore, promotes RANKL expression and bone resorption in association with upregualtion of the IL-17 effector, Act1, supporting the concept that post-menopausal osteoporosis is a disorder of innate immunity.