CONTROL OF OSTEOCLAST ACTIVATION AND DIFFERENTIATION

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Osteoclast bone resorption is the first step in bone remodeling, and can be regulated at three distinct steps: osteoclast differentiation, initiation of bone resorption, and control of the resorption rate (or duration). Osteoclasts resorb bone by adhering to bone matrix proteins, and forming an enclosed space at the attachment site into which proteolytic enzymes are secreted, and protons are actively transported by a vacuolar H+-ATPase (V-ATPase) that is highly amplified in the ruffled membrane. Recent studies from our laboratory suggest that expression of the V-ATPase 15 kD subunit may have a key role in regulating this subunit expression in a model for osteoclast differentiation that is dependent on cell-cell interactions. Immunoprecipitation and immunoblotting will be performed marrow cultured to determine if the 15 kD subunit controls the overall amount of functional V-ATPase. The mechanism of induction of 15 kD subunit expression will be studied by stable transduction of promoter- reporter constructs into a murine monocytic precursor line that differentiates into osteoclast-like cells in co-culture. In studies on the initiation of bone resorption in mouse marrow cultures, we discovered that interstitial collagenase (the homolog of human MMP-13), produce by stromal cells, has a key role in initiating bone resorption that does not involve clearing the bone surface of collagen, by production of collagen cleavage products that activate bone resorption. An objective of this project will be to determine if collagen cleavage products activate isolated osteoclasts directly; to examine the enzymatic and cell biologic mechanisms by which MMP-13 initiates osteoclast bone resorption by determining the site(s) on collagen and structural requirements for induction of osteoclast bone resorptive activity using recombinant collagen proteins and in vitro bone-resorption assay, in both murine and human cell models. These studies should enhance our understanding of the regulation of normal bone remodeling, and may provide new insights on derangements that occur in metabolic bone diseases.