The specific aims of this proposal are 1) to analyze in detail the chemistry and biosynthesis of bone proteoglycans, 2) to use antibodies in immunolocalization experiments, and 3) to evaluate the molecular mechanism by which 1,25-dihydroxyvitamin D3 appears to regulate osteopontin biosynthesis. The first two aims depend on classical biochemical and immunological methods while the last aim relies, to some extent, on the techniques of molecular biology. The applicant has some experience using these techniques, however, the funding of this Research Career Development Award would greatly enhance his research career development by allowing more extensive training in this area. The major hypothesis upon which this research is based is that non-collagenous components of mineralized connective tissues are intimately involved in the formation of the tissues. Furthermore, an understanding of the chemial composition, localization and regulatory mechanisms of biosynthesis are required before specific, functional roles can be elucidated. In this proposal we will address the aforementioned areas as they relate to proteoglycans and osteopontin. The approach taken is to isolate chemical amounts of these components, verify their purity and use them as antigens. The antibodies will be used in immunolocalization studies and as reagents in immunoassays. We will explore the kinetics of proteoglycan biosynthesis in rat bone, in ROS 17/2.8 cells and in cultures of rat calvarial osteoblasts. We will evaluate the role and mechanism that 1,25- dihydroxyvitamin D3 may have in the regulation of osteopontin biosynthesis.
