This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Bone resorption is mediated by a mechanism in which local acidification of the osteoclast-bone interface depends on a vacuolar type H+ATPase charged coupled to the CLC-7 chloride channel. The acidified microenvironment first mobilizes the mineral phase of bone followed by organic matrix digestion via the lysosomal cysteine protease, Cathepsin K. Although the H+ATPase, CLC-7 and cathepsin K are critical to bone resorption, little is known about how they are targeted to the cell's polarized ruffled membrane. Compelling evidence indicates that these three bone resorptive moieties are localized in lysosomes in non-resorbing osteoclasts and are transported to the ruffled border when the cells are activated. Thus, characterizing the molecular mechanisms of the targeted secretion of lysosomes in osteoclasts will promote our understanding of how they degrade bone. With this in mind, we have isolated a lysosome-like vesicular fraction from mature osteoclasts, which contains cathepsin K and LAMP2 (Igp110), a lysosome-associated membrane protein. Separately, we find that targeted disruption in mice of synaptotagmin VII (syt VII), a protein that mediates secretion of lysosomal contents in fibroblasts and the neuro-endocrine cells , inhibits cathepsin K secretion by osteoclasts and bone resorption in vitro and in vivo. Given that 1) cathepsin K is secreted into the resorptive microenvironment of osteoclasts and LAMP2 is localized at the ruffled border of resorbing osteoclasts and 2) syt VII regulates lysosome secretion in fibroblasts and neuroendocrine cells, we hypothesize that osteoclastic bone resorption is mediated by exocytosis of lysosomal-like vesicles, via a mechanism involving sytVII. Thus, our specific aims are to: 1) identify the molecular machinery regulating lysosome trafficking and secretion by detailed proteomic analysis of osteoclast lysosomes and 2) define the mechanisms by which syt VII regulates lysosome secretion and osteoclast function by identifying its binding proteins in osteoclasts.
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