Research Project
3159807
We plan to isolate and characterize the factor(s) through which osteoclasts stimulate osteoclastic resorption by analysis of supernatants using the isolated osteoclast resorption assay. We will identify the responsiveness of osteoclasts to cytokines and growth factors, and other known and potential regulators of bone resorption. The role of second messengers in the regulation of bone resorption by osteoclasts will be assessed, and we will identify those matrix components in bone that mediate adhesion and modulate bone resorption by osteoclasts. The lineage and regulation of differentiation of the osteoclast will be analyzed using the primary function of osteoclasts, the excavation of bone, supported by the osteoclast-specific monoclonal antibodies we have developed, as criteria for osteoclast-differentiation. Osteoclast lineage will be identified in semi-solid cultures of bone marrow cells. The effects of calcium-regulating hormones, cytokines and osteoblastic cells on osteoclastic development in colonies will be assessed. We will attempt to identify circulating precursors of osteoclasts in normal individuals and in patients and laboratory animals with increased levels of osteolysis by immunocytochemistry and the ability to excavate bone slices. We will identify leukemias that consist entirely or partially of osteoclastic precursors, using both fresh peripheral blood cells, and cells incubated under circumstances that induce osteoclastic differentiation. We will develop osteoclastic cell lines, since these could make a major contribution to analysis of the regulation of osteoclastic function. We propose to exploit our access to osteoclastomas. These tumours provide an opportunity to identify the osteoblastic phenotype associated with osteoclastic stimulation. We will raise monoclonal antibodies to the osteoblastic cells in these tumors, and develop cell lines which will further enable characterization of this phenotype. We propose to use osteoclasts from these tumors, as the only practicable source of human osteoclasts, to test the responsiveness of human osteoclasts, particularly to cytokines and growth factors that show species specificity. The tumors also represent a bulk of osteoclasts otherwise unavailable in mammals, from which we propose to purify and characterize the enzymes that resorb bone, with a view to identification of characteristics that may be amenable to therapeutic intervention, and to the development of novel markers for the measurement of resorptive activity in vitro and in vivo.
