Regulation of osteoclast motility and signalling

Resorption of mineralized tissues is a complex process which includes the migration of osteoclast precursors, fusion of precursors to form multinucleated osteoclasts, activation of resorption, migration of osteoclasts to new sites of resorption, and apoptosis. How paracrine factors regulate osteoclast migration and activity is largely unknown. We used time-lapse videomicroscopy to investigate the role of paracrine factors in regulating osteoclast motility, and fluorescence microspectrophotometry to study Ca2+ signalling. Both macrophage colony-stimulating factor (M-CSF) and transforming growth factor-beta (TGF-beta) induced membrane ruffling and chemotaxis of rat osteoclasts. These processes were inhibited by blockers of phosphatidylinositol 3-kinase (PI 3-kinase). In contrast, blockade of mitogen-activated protein (MAP) kinase kinases inhibited chemotaxis, but not membrane ruffling. These findings implicate PI 3-kinase and MAP kinase signalling pathways in the regulation of osteoclast motility. The alphavbeta3 integrin blocker, echistatin, caused osteoclast retraction and selectively inhibited chemotaxis of osteoclasts towards M-CSF, but not towards TGF-beta. In contrast, a blocking antibody against beta1 integrins selectively inhibited chemotaxis of osteoclasts towards TGF-beta, but not towards M-CSF. These data indicate the selective use of integrins by osteoclasts migrating in response to different chemotaxins. Whereas M-CSF and TGF-beta cause cytoskeletal remodelling leading to membrane ruffling and chemotaxis, the paracrine factor RANK ligand (RANKL) causes cytoskeletal remodelling leading to formation of actin rings. We found that RANKL also induces rapid transient elevation of cytosolic free Ca2+ concentration ([Ca2+];) in rat osteoclasts. These elevations of [Ca 2+]i persisted in the absence of extracellular Ca2+ and were abolished by inhibition of phospholipase C, indicating that RANKL induces release of Ca2+ from intracellular stores. RANKL-induced elevation of [Ca2+]i is a heretofore unrecognized signalling event, which may contribute to regulation of actin reorganization and osteoclast activation. These studies provide the first evidence that bone-derived paracrine factors are chemotaxins for osteoclasts, possibly inducing the migration to sites of physiological or pathological bone resorption. Our data suggest also that suppression of bone resorption in vivo by PI 3-kinase inhibitors and integrin blockers may be mediated, in part, through inhibition of osteoclast chemotaxis. The MAP kinase and Ca2+ signalling pathways may be additional targets for the development of antiresorptive drugs.