| The osteoclast is the exclusive cell responsible for bone resorption, making it a therapeutic target for treating skeletal disease. The cells resorptive ability depends upon the creation of an isolated acidic microenvironment at its interface with bone. This event requires the osteoclast to reorganize its cytoskeleton into an actin ring structure, a process requiring the signaling pathways emanating from the alphavbeta3 integrin and the M-CSF receptor, c-Fms. The cytoskeletal effector protein, Vav3, which is the guanine nucleotide exchange factor for the small GTPase, Rac, is also necessary for sealing zone formation. In this study we investigated the hypothesis that the adaptor protein SLP-76, which contains a binding site for Vav3, is involved in cytoskeletal rearrangements in the osteoclast. In addition to defining the role of SLP-76 in osteoclast function, we also delineated the pathway for alphavbeta3 and c-Fms-mediated SLP-76 activation via the Syk tyrosine kinase and the immunoreceptor activation motif (ITAM)-containing adaptor protein, DAP12. Lastly, we established the role of SLP-76 in the osteoclast and identified the structural components of SLP-76 that mediate its function.;Using osteoclasts derived from mice genetically deficient for Syk, DAP12, SLP-76, or the SLP-family member BLNK, we defined the c-Fms and alphavbeta3 signaling integrin pathways, both functionally and biochemically. alphavbeta3 and c-Fms engagement activates c-Src tyrosine kinase, which phosphorylates DAP12. DAP12 recruits Syk and promotes its activation, either by autophosphorylation after c-Fms ligation or phosphorylation by c-Src after alphavbeta3 occupancy. Both events converge on Syk-mediated SLP-76 and BLNK phosphorylation, which regulate osteoclast spreading and function. Accordingly, absence of SLP-76 abrogates Vav3 activation, while cytoskeletal remodeling is disrupted in cells expressing SLP-76 containing a mutation in the Vav binding site. Moreover, SLP-76 regulates osteoclast function in vivo. Mice lacking hematopoietic expression of SLP-76 or SLP-76 in combination with BLNK, fail to induce osteoclastic bone resorption following PTH injections compared to wild-type or those lacking BLNK alone, indicating that SLP-76 is the dominant adaptor in vivo. Collectively, in this series of studies, we demonstrate that SLP-76 regulates osteoclast function by linking Syk to Vav3 and is a potential target in the treatment of bone disease. |
