Bone is not essential for osteoclast activation.

BACKGROUND: The mechanism whereby bone activates resorptive behavior in osteoclasts, the cells that resorb bone, is unknown. It is known that α(v)ß(3) ligands are important, because blockade of α(v)ß(3) receptor signaling inhibits bone resorption, but this might be through inhibition of adhesion or migration rather than resorption itself. Nor is it known whether α(v)ß(3) ligands are sufficient for resorption the consensus is that bone mineral is essential for the recognition of bone as the substrate appropriate for resorption. METHODOLOGY/PRINCIPAL FINDINGS: Vitronectin- but not fibronectin-coated coverslips induced murine osteoclasts to secrete tartrate-resistant acid phosphatase, as they do on bone. Osteoclasts incubated on vitronectin, unlike fibronectin, formed podosome belts on glass coverslips, and these were modulated by resorption-regulating cytokines. Podosome belts formed on vitronectin-coated surfaces whether the substrates were rough or smooth, rigid or flexible. We developed a novel approach whereby the substrate-apposed surface of cells can be visualized in the scanning electron microscope. With this approach, supported by transmission electron microscopy, we found that osteoclasts on vitronectin-coated surfaces show ruffled borders and clear zones characteristic of resorbing osteoclasts. Ruffles were obscured by a film if cells were incubated in the cathepsin inhibitor E64, suggesting that removal of the film represents substrate-degrading behavior. Analogously, osteoclasts formed resorption-like trails on vitronectin-coated substrates. Like bone resorption, these trails were dependent upon resorbogenic cytokines and were inhibited by E64. Bone mineral induced actin rings and surface excavation only if first coated with vitronectin. Fibronectin could not substitute in any of these activities, despite enabling adhesion and cell spreading. CONCLUSIONS/SIGNIFICANCE: Our results show that ligands α(v)ß(3) are not only necessary but sufficient for the induction of resorptive behavior in osteoclasts; and suggest that bone is recognized through its affinity for these ligands, rather than by its mechanical or topographical attributes, or through a putative 'mineral receptor'.

Cathepsin K inhibitors prevent matrix-derived growth factor degradation by human osteoclasts.

The coupling between bone formation and resorption creates a therapeutic impasse in osteoporosis: antiresorptive therapy halts bone loss, but also inhibits bone formation, and therefore does not cure the condition. Surprisingly, recent preliminary reports suggest that inhibition of resorption by cathepsin K (CathK) inhibitors augments bone formation. Uniquely amongst resorption-inhibitors, CathK-inhibitors suppress degradation of the organic matrix of bone while allowing demineralization. We hypothesized that these unique characteristics might explain a capacity of CathK inhibitors to enhance bone formation: the inhibitors might prevent degradation not only of collagen, but also other proteins, including growth factors embedded in matrix. We tested this hypothesis using osteocalcin and insulin-like growth factor I (IGF-I) as examples of matrix-embedded proteins, and found that CathK-inhibitors, unlike other resorption-inhibitors, dramatically increased the concentrations of these matrix-derived proteins in supernatants of osteoclasts on bone, most likely through protection against intracellular degradation. We found that protons are both necessary and sufficient for the release of IGF-I from bone matrix, and that recombinant CathK can degrade both marker proteins. In the presence of a CathK-inhibitor, the amount of IGF-I released from matrix substantially exceeded the amount secreted by osteoclasts. CathK-inhibition similarly augmented bone morphogenetic protein (BMP)-2 release. Lastly, MC3T3-E1 numbers were greater after co-culture with osteoclasts on bone with versus without CathK-inhibitor, showing that, in the presence of CathK-inhibitor, osteoclasts release biologically-significant quantities of biologically-active matrix-derived growth factors. These results support a model in which osteoclastic secretion of protons demineralizes bone, causing release of growth factors from bone matrix. Normally these are largely degraded, with collagen, in the resorptive hemivacuole and during transcytosis to the basal surface of the osteoclast, but in the presence of CathK inhibitor they are released intact, and so might augment bone formation.