The three objectives of this investigation are to understand the role of MGP in preventing calcification of growth plate cartilage, to establish the role of MGP as a calcification inhibitor in mediating the action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on bone, and to determine the molecular mechanism by which MGP inhibits calcification. These studies are based solidly on the evidence that MGP is the in vivo inhibitor of cartilage calcification, evidence which has come from studies of the MGP gene knock-out mouse, and the warfarin-treated rat. The applicants' first Aim is to study the role of MGP in growth plate cartilage calcification in the rat. They will determine the first sites of warfarin-induced abnormal calcification in the growth plate of the proximal tibia in order to establish the points at which MGP normally acts to prevent cartilage calcification. They will also identify the mechanisms by which the activity of MGP is reduced in areas of normal growth plate mineralization; these studies include measuring the level of MGP expression in chondrocytes in normal areas of calcification, and investigating the post-translational modifications of MGP which are used to inactivate the protein at normal calcification sites.
Their second Aim i s to determine the role of MGP as a mediator of the action of 1,25(OH)2D3 on bone. They have recently found that 1,25(OH)2D3 causes a 35% decrease in the uptake of 45[Ca] into the epiphysis and metaphysis of the proximal tibia and that warfarin treatment completely blocks this decrease. They will further examine this effect in order to establish the role of MGP as a calcification inhibitor in this action of vitamin D on bone. They will also test the ability of MGP infusion to reduce the rate of mineralization in the proximal tibia. Finally, they will establish the role of MGP as a mediator of the action of 1,25(OH)2D3 on bone dietary calcium deficiency.
Their third Aim i s to carry out in vitro studies on the mechanism by which MGP inhibits calcification. They will use the recently discovered soluble form of MGP to investigate calcium and phosphate binding to the protein, to study the structure of the protein, and to investigate its interactions with apatite isolated from bone. They will also investigate the mechanism by which MGP inhibits calcification in vitro. The investigators will further study the demonstrated ability of MGP to inhibit calcification in supersaturated solutions, and will investigate the ability of MGP to inhibit matrix vesicle -initiated calcification.
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