The long-term objectives of this proposal are to develop an understanding of the progression of osteoporosis and to develop a therapy for osteoporosis which acts by blocking cell adhesion. This will be accomplished by identifying relevant adhesion receptors present in osteoblasts and osteoclasts and examining their biochemical and cellular regulation using a series of adhesion antagonists we have developed. The initial goal will be to measure the binding of bone adhesive proteins osteopontin, bone sialoprotein and thrombospondin to two closely related adhesion receptors, integrins a, b, and a,b,. The ligand association and dissociation rate constants will be determined in purified ligand- receptor binding assays and with surface plasmon resonance. Importantly, the binding constants will be carefully examined under extremes of [Ca2+] and pH, both of which are found at the bone surface during resorption by osteoclasts. To determine the physiological relevance of the binding data between these ligands and receptors, both will be localized in bone sections with immunohistochemistry using highly specific polyclonal and monoclonal antibodies. Integrins alphavBeta3 and alphavBeta5 are closely related in structure. However, major functional differences between the two are apparent. It is still not evident what the adhesion of cell lines lacking either alphavBeta3 or alphavBeta5 to bone matrix proteins. As an extension of these studies, the contribution of each receptor to osteoclast and osteoblast adhesion will be determined. Ultimately, this information will bear directly on the design of therapy for osteoporosis because the treatment should target osteoclast adhesion without affecting osteoblasts. Osteoclasts are known to undergo morphological changes as a result of changes in ion fluxes. It is hypothesized that binding of adhesion proteins can generate signals leading to these changes. Therefore, another goal is to determine which adhesion receptor, alphavBeta3 or alphavBeta5, transmit intracellular Ca2+ fluxes across cell membranes in response to specific ligands for each receptor. The changes in Ca2+ flux will be measured when ligand is immobilized, and acting like extracellular matrix, and when the ligand is soluble. Finally, the central hypothesis of our proposed studies is that a precisely directed blockade of osteoclast adhesion to bone can be used to halt osteoporosis. This hypothesis will be tested with highly specific antagonists that we have developed against adhesion receptors in bone. As a first, step, these antagonists will be tested for the ability to interfere with osteoclast-mediated resorption of bone particle in vitro. Ultimately, these antagonists will be used to challenge osteoporosis in a rat model.
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