The overall goal of this proposal is to develop new experimental strategies to elucidate the functional roles of the bone matrix molecules, osteocalcin (OC) and bone sialprotein (BSP) during bone tissue growth and remodeling. We propose (1) to develop transgenic osteoblast cell lines that constitutively express normal and site-directed mutants of OC and BSP and (2) to develop in vitro and in vivo procedures to assess the precise functional roles that OC and BSP play in bone mineralization and remodeling using these transgenic cells. Experiments have been designed to critically test the following two hypotheses: that OC provides signals within the extracellular matrix (ECM) that promote osteoclastogenesis and/or bone resorption and that BSP provides positional information within the ECM that is required for osteoblastic and osteoclastic interaction with the mineralized phase. High efficiency plasmid and retroviral vectors will be developed for the transfection and selection of avian OC and BSP genes within specific osteoblastic cell lines. Use of avian genes offers the unique advantage that sequence differences between the avian genes and their murine counterparts provides a means to identify with both molecular and antibody probes the endogenous mouse from the avian gene products. Structure/function relationships of OC and BSP on extracellular matrix assembly, mineralization, and osteoblast interaction with, or response to the ECM will be determined. In vitro studies will focus on ECM formation and mineralization of the cell lines grown in culture under mineralizing conditions, while preparative scale levels of the recombinant mutant proteins will be prepared for screening of their role in promoting cell adherence and phenotypic effects on cultured osteoclasts and osteoblasts. Parallel experiments will examine the structure/function relationships of OC and BSP expression within ECM on osteoclast function and osteoclastogenesis. Both in vivo and in vitro approaches will be used in these studies. For the in vivo studies the transgenic cells will be grown in novel three-dimensional culture devices followed by implantation into normal syngeneic mice. Histomorphometric parameters will be compared to determine the effects of the expression of the specific transgenes on the stoichiometric accumulation of specific ECM proteins and mineral within the tissue. For studies on remodeling the transgenic tissues which develop in vivo will be examined by biochemical, histomorphometric, in situ hybridization, and molecular analysis of osteoclastic development and functional bone resorption.
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