pyrophosphate and monodansylcadaverine

Calcium pyrophosphate dihydrate (CPPD) crystals are commonly found in osteoarthritic joint tissues, where they predict severe disease. Unlike other types of calcium phosphate crystals, CPPD crystals form almost exclusively in the pericellular matrix of damaged articular cartilage, suggesting a key role for the extracellular matrix milieu in their development. Osteopontin is a matricellular protein found in increased quantities in the pericellular matrix of osteoarthritic cartilage. Osteopontin modulates the formation of calcium-containing crystals in many settings. We show here that osteopontin stimulates ATP-induced CPPD crystal formation by chondrocytes in vitro. This effect is augmented by osteopontin's incorporation into extracellular matrix by transglutaminase enzymes, is only modestly affected by its phosphorylation state, and is inhibited by integrin blockers. Surprisingly, osteopontin stimulates transglutaminase activity in cultured chondrocytes in a dose-responsive manner. As elevated levels of transglutaminase activity promote extracellular matrix changes that permit CPPD crystal formation, this is one possible mechanism of action. We demonstrate the presence of osteopontin in the pericellular matrix of chondrocytes adjacent to CPPD deposits and near active transglutaminases. Thus, osteopontin may play an important role in facilitating CPPD crystal formation in articular cartilage.

Topics: Adenosine Triphosphate; Animals; Cadaverine; Calcinosis; Calcium Pyrophosphate; Cartilage, Articular; Chondrocytes; Cystamine; Diphosphates; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Immunohistochemistry; Osteopontin; Sus scrofa; Thrombin; Transglutaminases

Transglutaminases (TGases) (E.C. 2.3.2.13) catalyze a posttranslational modification of proteins and are associated with biomineralization in growth plate cartilage. Type II TGase participates in the activation of latent transforming growth factor beta (TGFbeta), a crucial factor for both normal cartilage mineralization and the pathologic mineralization that results in calcium pyrophosphate dihydrate (CPPD) crystal formation in aging articular cartilage. To explore a possible association between TGase levels and CPPD crystal formation in mature articular cartilage, TGase activity in articular chondrocytes from old and young pigs and in the articular cartilage vesicle (ACV) fraction of porcine articular cartilage was examined. In addition, the effects of TGase inhibitors on the production of inorganic pyrophosphate (PPi), a process necessary for CPPD crystallogenesis, were determined.. TGase activity was measured with a radiometric assay in cultured articular chondrocytes from the knee joints of old (3-5 years old) and young (2-6 weeks old) pigs and in the ACVs. PPi levels were measured in chondrocyte-conditioned media in the presence of TGase inhibitors or control compounds.. Levels of TGase activity in the cytosolic fraction of old chondrocytes were 7-fold higher than those in identically cultured young chondrocytes. The mean +/- SD activity level in the membrane fraction of lysed chondrocytes was 6.0 +/- 0.6 units/mg protein in old articular chondrocytes and was undetectable in young chondrocytes. In ACVs, the mean +/- SD TGase activity level was 1.23 +/- 0.1 units/mg protein. Type II TGase protein was present in chondrocyte cytosol and in ACVs. TGase activity was increased by TGFbeta to 120% of control values (P < 0.01), and decreased by insulin-like growth factor 1 to 80% of control values (P < 0.01). TGase inhibitors blocked media accumulation of PPi, an essential precursor of CPPD crystal formation, and a sensitive marker of TGFbeta effect.. These data suggest a potential link between TGase activity and processes of pathologic biomineralization that result in CPPD crystal formation in aging articular cartilage.

Topics: Aging; Aminoacetonitrile; Animals; Cadaverine; Cartilage, Articular; Cystamine; Diphosphates; Enzyme Inhibitors; Growth Substances; Hydrolysis; Methylhistamines; Swine; Transglutaminases