Chondrocyte death by apoptosis is associated with the initiation and severity of articular cartilage degradation.

AIM: To investigate the role of chondrocyte apoptosis in the initiation and severity of articular cartilage (AC) damage. METHODS: Articular cartilage from equine metacarpophalangeal (MCP) (n = 13) and metatarsophalangeal (MTP) (n = 16) joints was used and each graded macroscopically for cartilage degradation (macroscopic osteoarthritis [OA] grade). Cartilage was sampled from six regions on the articular surface of both joint types and graded using a 'modified' Mankin scoring system. Apoptosis of chondrocytes in cartilage sections was assessed by expression of active caspase-3 using indirect immunohistochemistry. RESULTS: Apoptosis was found to increase significantly with macroscopic OA grade (P < 0.0001). There was a significant trend for increasing 'modified' Mankin score with increasing macroscopic OA grade (P < 0.0009). Apoptosis was significantly higher in the superficial zone than in the middle or deep zones (P < 0.01 and P < 0.001, respectively). The incidence of apoptosis correlated significantly with the early stages of microscopic cartilage damage ('modified' Mankin scores 0-3). Significant differences in overall apoptosis were noted when cartilage specimens with a 'modified' Mankin score of 3 were compared to grade 2 (P < 0.001), grade 1 (P < 0.001) and grade 0 (P < 0.05) specimens. However, no significant difference in overall apoptosis was noted between grade 3, 4 and 5 samples. CONCLUSIONS: The positive correlations of chondrocyte apoptosis with early stages of OA and severity of cartilage damage in the joints, suggest that this process is intrinsically linked to cartilage damage and may be associated with the initiation of cartilage degradation in OA.

Transglutaminase factor XIIIA in the cartilage of developing avian long bones.

Previously, we showed that mRNA for transglutaminase factor XIIIA (FXIIIA) is up-regulated in the hypertrophic zone of the growth plate of the chicken tibiotarsus, a well-characterized model of long bone development. In the present study, we have studied the distribution of the FXIIIA protein and of transglutaminase enzymatic activity in this growth plate, as well as in the cartilage of the epiphysis, which includes that of the articular surface. By immunohistochemical analysis, the protein is detected in the zone of maturation, where it is mostly intracellular, and in the hypertrophic zone, where it is present both intracellularly and in the extracellular matrix. The intracellular enzyme is mostly a zymogen, as determined with an antibody specific for the activation peptide. Externalization of FXIIIA is accompanied by enzyme activation. To study the pattern of transglutaminase activity, a synthetic transglutaminase substrate, rhodamine-conjugated tetrapeptide (Pro-Val-Lys-Gly), was used for pulse labeling in organ cultures. Intensive incorporation of the fluorescent substrate was observed throughout the hypertrophic zone and in the cells surrounding the forming blood vessels. The patterns of FXIIIA immunostaining and substrate incorporation overlap almost completely. The cartilaginous factor XIIIA is different from the plasma form in that, both intracellularly and extracellularly, it exists as a monomer, as determined by Western analysis, whereas the plasma form of FXIII is a tetrameric complex composed of both A and B subunits. We also identified FXIIIA and transglutaminase activity within the articular and condylar regions of the tarsus, suggesting a possible involvement of mechanical pressure and/or stress in the production of the molecule and subsequent cross-linking of the cartilage matrix. Thus, transglutaminases, in particular FXIIIA, are involved in the formation of long bones through its activity both in the hypertrophic region of the growth plate and in the formation of articular/epiphyseal cartilages.

Secretion of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from cultured chick embryo chondrocytes.

We found that chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase were released into the culture medium from the cultured chick embryo chondrocytes. Since the release of the sulfotransferases was observed not only in serum-supplemented medium but also in serum-free medium, the released sulfotransferases were unlikely to be derived from serum. Addition of ascorbate to the serum-free medium supported the continuous release of the sulfotransferases. Monensin, which is known to cause dilatation of the Golgi apparatus and to inhibit sulfation of proteoglycan, was found to affect the release of the sulfotransferases. In the presence of 10(-6) M monensin, chondroitin 6-sulfotransferase activity in the cell layer was decreased to less than one tenth of the control, and the rate of the release of the activity became much smaller than the control after the initial rapid release. The activity of chondroitin 4-sulfotransferase was also affected by monensin, but the reduction of the chondroitin 4-sulfotransferase activity in the cell layer was not so great as the reduction of chondroitin 6-sulfotransferase activity. Unlike to the microsomal sulfotransferases, both chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase released into the culture medium were retained in the soluble fraction after centrifugation at 100,000 x g for 60 min, and were not activated by detergent. pH optimum and requirements for sulfhydryl compounds of the released sulfotransferases were similar to those observed previously in the chondroitin sulfotransferases from chick embryo cartilage and from cultured chick embryo chondrocytes. These results suggest that chondroitin sulfotransferases, which are localized in the Golgi apparatus, may be secreted to the extracellular space in a soluble form under the culture conditions.