The overall goal of this program project is (he elucidation of the metabolism and mechanism of action of fat-soluble vitamins A, D and K. A primary goal is to elucidate the molecular mechanism of action of 1,25- dihydroxyvitamin D3 (1,25-(OH)2D3). This will include the generation of large amounts of receptor and determination of its 3-dimensional structure using methods such as NMR spectroscopy and X-ray crystallography. An in vitro transcription system will be developed for the isolation and identification of all factors required for the vitamin D-induced gene expression system. The role of phosphorylation of the vitamin D receptor in transcription will be studied. The vitamin D receptor gene will be completely elucidated including the promoter region and the regulation thereof. The vitamin D 1alpha-hydroxylase will be purified and used to generate monoclonal antibodies and ultimately cloned. The molecular mechanism of regulation of the 1alpha-hydroxylase and 24-hydroxylase will be fully emphasized. The molecular mechanisms whereby 1,25-(OH)2D3 causes the in vivo mobilization of calcium from bone to plasma will be investigated at the physiologic level using osteopetrotic mice possessing genetic defects in the osteoclastic system and in vitro using osteoblast and osteoclast cultures. The functional form of vitamin A required for female reproduction will be determined especially that prevents fetal resorption in retinoic acid-supported rats and its mechanism studied. Retinoic acid receptor gamma will be produced in large amounts from a baculovirus system in preparation for X-ray crystallography and NMR to determine its 3-dimensional structure. A determination of precisely when retinoic acid is needed in vivo for normal embryonic development, especially the nervous system, will be made and in primary cultures determine how retinoic acid functions to promote neuronal differentiation and survival. The mechanism of action and substrate specificity of the vitamin K-dependent carboxylase will be studied. The differential response of prothrombin and FX biosynthesis to warfarin will be investigated in rat and human cells. A cellular vitamin K binding protein will be isolated, and a postulated conversion of phylloquinone to menaquinone-4 will be investigated in chick liver. The influence of vitamin K status on skeletal health will be studied in a rat model, and a vitamin K/vitamin D interaction in the gamma-carboxylation of the bone protein osteocalcin will be investigated.
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