This renewal application focuses on studies to define the regulation of synthesis of the vitamin K dependent bone protein, osteocalcin by three factors which we believe are key to understanding its function. 1) the role of the pro-piece of osteocalcin in regulation of osteocalcin secretion, cleavage and Gla synthesis; 2) the precise function of Gla residue synthesis in osteocalcin and the requirements for vitamin K utilization in bone cells for Gla synthesis; and 3) specific definition of the induction of osteocalcin synthesis by calcitriol as related solely to cellular level of transcripts and/or to accelerated processing via the carboxylase enzyme and functionally to bone resorption. To achieve these objectives, specific questions of investigation are the following: a) the site (e.g., Golgi, microsomes, extracellular) of propiece cleavage and the mechanism (e.g., serine protease) of cleavage will be investigated; b) prove that the propiece is glycosylated and is a signal for secretion; c) define the effect of warfarin on the bone vitamin K cycle, intracellular precursor accumulation and osteocalcin peptide synthesis; d) determine the requirement of and precise number of Gla residues for pro- osteocalcin cleavage and osteocalcin function. The majority of the proposed studies will be carried our in a newly developed chick osteoblast culture system in which second passage cells (greater than 90% pure osteoblasts) undergo a programmed differentiation throughout a 3 week culture period producing a matrix that calcifies even in the absence of a chemical inductant. In this system pro-osteocalcin and osteocalcin function can be addressed by examining alterations in matrix formation, mineral deposition and bone cell activity (measuring parameters of alkaline phosphatase and collagen synthesis) that result from 1) inhibition of propeptide cleavage; 2) inhibition of pro-osteocalcin secretion; 3) inhibition of Gla synthesis in the protein by warfarin; and 4) the induction of osteocalcin synthesis by calcitriol. Utilizing a cDNA probe of osteocalcin, mRNA levels and activity will be quantitated as affected by warfarin and calcitriol and how inhibition of these regulative factors effect bone cell activity and calcified matrix formation, will allow us to better define its function and thereby provide a more meaningful parameter of osteocalcin measurements in a clinical setting.
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