Osteocalcin, the major noncollagenous bone protein, characterized by the vitamin K-dependent calcium-binding amino acid, Gamma-carboxyglutamic acid (G1a) has well-defined molecular and physical properties. Recently, it has been found that bone that is 99% deficient in osteocalcin and 93% deficient in Gla is poorly resorbed both in vivo, when implanted subcutaneously in a rat model, and in vitro, when incubated with human monocytes. These observations agree with previous studies implicating osteocalcin in calcium regulation. For example, the chemotactic property of osteocalcin for cells capable of resorbing bone and the specific increase in osteocalcin synthesis by 1,25(OH)2D3, a hormone which can promote bone resorption. This proposal will investigate the precise mechanism for the observed defect in the decreased resorption of osteocalcin-deficient bone. Bone from the warfarin-treated and K-deficient rat wil be chemically characterized for other protein alterations (analyses by 2D gels and HPLC profiles). The critical levels of osteocalcin and/or other Gla-containing proteins necessary to achieve the defect will be established. Experiments will compare the resorption of devitalized normal and osteocalcin-deficient bone in a model system of in vivo implant of mineralized bone particles (BP) into subcutaneous packets. In this model blood derived progenitor cells differentiate into osteoclasts that resorb control bone from 5 through 28 days after implantation. Specific cell types are identified morphologically and resorption is quantitated by hisotomorphometric analysis (number of multinucleated cells and percent mineralized matrix) and biochemically (calcium content, DNA and tartrate-resistant acid phosphatase activity). These studies will provide information on whether osteocalcin (or some other vitamin K-sensitive protein) is necessary for 1) recruitment of cells to bone; 2) attachment to mineralized surfaces; 3) promotes differentiation of a progenitor cell to a bone-resorbing cell; or 4) is involved in stimulation of activity of bone resorbing cells. The knowledge of cell-matrix interactions to be gained from the proposed studies has potential applications in defining the control mechanisms of normal bone resorption, in explaining the pathogenesis of metabolic bone diseases, inflammatory bone destruction (peridontal disease) and in pathogenesis and treatment of pathologic mineral deposition.
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