Increased chondrocyte PPi production, and PPi-generating NTPPH activity are linked with CPPD crystal deposition disease, common in aging and osteoarthritic cartilage. Because the major chondrocyte NTPPPH is PC-1, we will study the regulation of PC-1 expression and the primary role PC-1 could play in crystal deposition and cartilage degeneration. We will: 1. Define the mechanism of PPi elaboration by chondrocytes: To define if PPi elaboration is critically regulated by TGFbeta-regulated expression and movement to the membrane of PC- 1, we will determine the distribution of cellular, membrane and secreted Pc-1 and NTPPH when PPi release is induced (using TGFbeta-stimulated chondrocytes, and SV40-immortalize chondrocytes transfected with PC-1. Soluble, secreted PC-1 cDNA and enzyme- deficient PC-1 cDNA will serve as controls, and we will concurrently measure the NTPPH substrate ATP, PPi, and pyrophosphatase activity. To directly test the roles of PC-1 transcription and translocation in chondrocyte PPI release, we will use antisense PC-1 and brefeldin A, respectively. Last, we will define the mechanisms for the known abilities or IL-1beta and IGF-1 to inhibit TGFbeta- induced chondrocyte PPi elaboration. 2. Define the relationship between PC-1 expression and chondrocyte differentiation and growth factor responsiveness: We will determine how PC-1 expression is regulated in distinct stages of chondrocyte differentiation and function (TGFbeta-regulated cell cycle progression, proliferative senescence, hypertrophy, apoptosis, de-differentiation). Because PC-1 is a major inhibitor of the insulin receptor kinase in fibroblasts, we will test the hypothesis that PC-1 expression directly influences chondrocyte responsiveness to growth factors, including the highly homologous IGF-1 receptor, by transfecting immortalized chondrocytes with PC-1. 3. Define the effects of increased PC-1 expression on cartilage in transgenic mice: To test the hypothesis that increased PC-1 exerts primary effects on cartilage in vivo, we will study mice from lineages transgenic for PC-1/NTPPPH, under control of the type II collagen promoter-enhancer. Control lineages transgenic for enzyme-deficient PC-1 will be studied. Cartilage morphology, crystal deposition, PPi concentration, and PC-1 and NTPPPH expression will be studied in transgenics (and nontransgenic littermates) allowed to age (to 18 months), using histomorphometry, histochemistry, radiography, atomic force microscopy and X-Ray diffraction.
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