Transglutaminase contributes to CPPD crystal formation in osteoarthritis.

Calcium pyrophosphate dihydrate (CPPD) crystals are common components of osteoarthritic joints and correlate with a poor prognosis. Transglutaminase (Tgase) enzymes have been implicated in pathologic mineralization in cartilage; yet, definitive studies linking Tgase activity to CPPD crystal formation in osteoarthritic articular cartilage are lacking. We measured in-vivo Tgase activity in osteoarthritic and normal human cartilage, and explored the effect of Tgase inhibitors on CPPD crystal formation by normal chondrocytes. Osteoarthritic articular cartilage from was obtained from specimens discarded at the time of knee replacement surgery. Normal adult cartilage samples from a tissue bank were used as controls. Tgase-specific isopeptide (epsilon-(gamma-glutamyl) lysine) bonds were measured in cartilage extracts by HPLC. Tgase-specific crosslinks were localized in osteoarthritic cartilage by immunohistochemistry. The effect of Tgase inhibition was determined in an in-vitro model of CPPD crystal formation. Tgase-specific crosslink levels were 1.55 +/- 0.3 picomoles/ng protein in normal human adult articular cartilage and 4.74 +/- 0.7 picomoles/ng protein in osteoarthritic human cartilage (p less than 0.001). Immunostaining confirmed the presence of Tgase crosslinks in the pericellular matrix of chondrocytes at potential sites of CPPD crystal formation. Tgase inhibitors suppressed CPPD crystal formation by porcine chondrocytes. These findings support a role for Tgase in CPPD crystal formation in aging or degenerated cartilage.

Regulation of transglutaminase activity in articular chondrocytes through thrombin receptor-mediated factor XIII synthesis.

Transglutaminases are a family of enzymes that catalyze the formation of epsilon-(gamma-glutamyl)lysine isopeptide bonds in proteins, an activity that has been implicated in the pathogenesis of cartilage matrix mineralization in degenerative arthritis. Type II transglutaminase and thrombin-activatable factor XIII have been identified in articular cartilage. Thrombin, a coagulation protease, is found in pathological synovial fluids, and is known to stimulate transglutaminase activity in non-articular tissues. We investigated the effects of thrombin on transglutaminase activity in porcine articular chondrocytes. Direct addition of thrombin to chondrocyte lysates resulted in increased transglutaminase activity due to proteolytic conversion of factor XIII to XIIIa. Thrombin-treated chondrocyte cultures (0.001 to 2.0 U/ml) also showed increased transglutaminase activity. Thrombin treatment of chondrocyte cultures increased transglutaminase activity as early as 15 minutes after addition, an effect that we attributed to factor XIII activation. Additional stimulatory effects of thrombin were observed in cultured chondrocytes at 4 and 24 hours. A thrombin receptor agonist peptide (TRAP) which activates the PAR1 thrombin receptor mimicked these later effects. Thrombin treatment of chondrocyte cultures increased factor XIII mRNA and protein levels, without affecting levels of type II transglutaminase. Thus, thrombin stimulates transglutaminase activity in articular cartilage by directly cleaving factor XIII and by receptor-mediated up-regulation of factor XIII synthesis. Such increases in potential transglutaminase activity may facilitate pathological matrix calcification in degenerative arthritis.