This proposal uses a cartilage cell model to address how calcifying cells modulate events at distal sites in the extracellular matrix and in particular, on the role of matrix vesicles (MV) in this process. It is based on the hypothesis that vitamin D metabolites regulate MV composition via genomic mechanisms through the classic vitamin D receptor (VDR) and membrane-mediated pathways. In addition, chondrocytes synthesize and secrete vitamin D metabolites, as well as other autocoids like prostaglandin E2, which, in turn, interact directly with the MV membrane to nongenomically regulate MV activity.
Three specific aims are proposed. The first is to examine the biochemical mechanisms involved in the production of vitamin D metabolites by chondrocyte cultures. Regulation of l,25-(OH)2 D3 (1,25) and 24,25-(OH)2 D3 (24,25) production by 1,25 and 24,25 will be examined. Changes in 1-alpha- and 24-hydroxylases, as well as gene expression of the 24-hydroxylase, will be examined as a function of hormone dose and time. The role of the VDR or membrane-initiated events will be assessed using structural analogs of 1,25 and 24,25. The second is to examine the regulation of membrane signaling systems by Vitamin D metabolites. The mechanisms for differential regulation of protein kinase C (PKC) by 1,25 and 24,25 in plasma membranes (PM) and MV and use analogues of 1,25 and 24,25 to assess the relative contributions of the VDR, as well as the role of functional moieties of the vitamin D metabolites in their nongenomic action on MV membranes will be examined. The interrelationship between the PKC and PGE2 pathways will also be determined. The third is to to characterize the membrane effects of 1,25 and 24,25 on the functional properties of MV's. The regulation of stromelysin-1 by 1,25 and 24,25 will be examined, and will focus on the role of cell maturation state and the relative contributions of the VDR and membrane-mediated pathways in regulating stromelysin-1 synthesis, secretion, and activation. Nongenomic regulation of MV's will be assessed in functional assays of calcification in proteoglycan-containing gelatin gels. This work will have major implications for our understanding of MV and how cells that calcify their matrix are able to regulate these extracellular organelles at distant sites.
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