Bone development and remodeling are controlled in large part by factors which regulate the differentiation of osteoprogenitor cells into osteoblasts or which regulate the activity of osteoblasts themselves. This study is aimed at understanding at the molecular level the mechanisms which regulate phenotypic expression in osteoblasts. To approach this problem, the expression of alkaline phosphatase, a phenotypic marker for osteoblasts, will be examined in a permanent clonal osteoblastic rat osteosarcoma cell line, ROS 17/2.8. The specific goals of the study are to use a specific antiserum prepared against rat osteosarcoma alkaline phosphatase to (i) characterize the biosynthesis of the enzyme in ROS 17/2.8 and (ii) develop the means to produce a specific cDNA probe for alkaline phosphatase. First, the biosynthesis of alkaline phosphatase in ROS 17/2.8 cells will be measured and correlated with changes in enzyme specific activity which occur in culture. Second, PTH, 1,25(OH)2D3 and dexamethasone, three hormones shown to modulate alkaline phosphatase activity in bone and in cultured osteoblastic cells, will be examined to determine their specific effects on enzyme synthesis, as a step toward more precisely defining their mechanism(s) of action in osteoblastic cells. Third, using the above findings as a basis, the culture conditions permitting optimal synthesis of alkaline phosphatase will be determined. Fourth, selection techniques (e.g. fluorescence activated cell sorting) will be used to isolate sublines based on high alkaline phosphatase expression. Alkaline phosphatase mRNA will then be isolated from these """"""""high producer"""""""" cells by specific immunoabsorption of polyribosomes for synthesis of cDNA probe. These studies should aid in understanding the mechanisms of normal and pathologic bone metabolism.
