Bone non- collagenous proteins (NCPs) appear to be involved in several aspects of bone formation, as well as in the homeostatic mechanisms relative to bone remodeling. One bone NCP, osteopontin (OPN) is a sialic acid-rich phosphoprotein that is synthesized and secreted by osteoblasts and osteoclasts. A conserved RGDS sequence promotes cell attachment (osteoblasts and osteoclasts) and spreading, and possibly intracellular signaling, through interaction with a cell surface, alphav beta3 integrin. OPN may also influence the rate of mineral formation and the resultant crystal habit. The biosynthesis of OPN is up-regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]; thus, OPN may manifest the actions of this hormone in its effects on bone formation and mineral homeostasis. The investigators have recently shown that, after short-term exposure of cells to 1,25(OH)2D3, there is a change in charge state of secreted OPN, including an affect on phosphorylation. The applicants have also studied the genomic regulation of OPN biosynthesis by 1,25(OH)2D3 and have found 2 vitamin D response elements (VDRE) on rat OPN gene which are involved in the increased transcription, but are regulated differently. They hyposize that the regulation of osteoblastic synthesis of OPN by 1,25(OH)2D3 involves both transcriptional and non-transcriptional mechanisms. In order to address this hypothesis, the following Specific Aims are proposed: 1) to elucidate the location and nature of the post-translational modifications (PTM) of rat bone OPN; 2) to characterize the nature and mechanism of the non-transcriptional regulation of OPN biosynthesis and PTM changes brought about by incubation of cells with 1,25(OH)2D3; and 3) to elucidate the mechanisms of genomic regulation of OPN synthesis of 1,25(OH)2D3. These studies will be performed by an interdisciplinary team of investigators. The applicants hope that the results will increase our knowledge concerning the manner by which 1,25(OH)2D3 influences bone formation and remodeling.
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