B Noé1, A R Poole2, J S Mort2, H Richard3, G Beauchamp4, S Laverty5. 1. Comparative Orthopaedic Research Laboratory, Département de Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe, QC J2S 2M2, Canada. Electronic address: beatriz.noe@umontreal.ca. 2. Division of Orthopaedics, Department of Surgery, McGill University, Montreal, QC, Canada. 3. Comparative Orthopaedic Research Laboratory, Département de Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe, QC J2S 2M2, Canada. 4. Département de Pathologie et Microbiologie Vétérinaires, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe, QC J2S 2M2, Canada. 5. Comparative Orthopaedic Research Laboratory, Département de Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe, QC J2S 2M2, Canada. Electronic address: sheila.laverty@umontreal.ca.
Abstract
OBJECTIVES: Develop a species-specific ELISA for a neo-epitope generated by cathepsin K cleavage of equine type II collagen to: (1) measure cartilage type II collagen degradation by cathepsin K in vitro, (2) identify cytokines that upregulate cathepsin K expression and (3) compare cathepsin K with matrix metalloproteinase (MMP) collagenase activity in stimulated cartilage explants and freshly isolated normal and osteoarthritic (OA) articular cartilages. DESIGN: A new ELISA (C2K77) was developed and tested by measuring the activity of exogenous cathepsin K on equine articular cartilage explants. The ELISA was then employed to measure endogenous cathepsin K activity in cultured cartilage explants with or without stimulation by interleukin-1 beta (IL-1β), tumour necrosis-alpha (TNF-α), oncostatin M (OSM) and lipopolysaccharide (LPS). Cathepsin K activity in cartilage explants (control and osteoarthritic-OA) and freshly harvested cartilage (control and OA) was compared to that of MMPs employing C2K77 and C1,2C immunoassays. RESULTS: The addition of Cathepsin K to normal cartilage caused a significant increase (P < 0.01) in the C2K77 epitope release. Whereas the content of C1,2C, that reflects MMP collagenase activity, was increased in media by the addition to cartilage explants of TNF-α and OSM (P < 0.0001) or IL-1β and OSM (P = 0.002), no change was observed in C2K77 which also unchanged in OA cartilages compared to normal. CONCLUSIONS: The ELISA C2K77 measured the activity of cathepsin K in equine cartilage which was unchanged in OA cartilage. Cytokines that upregulate MMP collagenase activity had no effect on endogenous cathepsin K activity, suggesting a different activation mechanism that requires further study.
OBJECTIVES: Develop a species-specific ELISA for a neo-epitope generated by cathepsin K cleavage of equinetype II collagen to: (1) measure cartilage type II collagen degradation by cathepsin K in vitro, (2) identify cytokines that upregulate cathepsin K expression and (3) compare cathepsin K with matrix metalloproteinase (MMP) collagenase activity in stimulated cartilage explants and freshly isolated normal and osteoarthritic (OA) articular cartilages. DESIGN: A new ELISA (C2K77) was developed and tested by measuring the activity of exogenous cathepsin K on equinearticular cartilage explants. The ELISA was then employed to measure endogenous cathepsin K activity in cultured cartilage explants with or without stimulation by interleukin-1 beta (IL-1β), tumour necrosis-alpha (TNF-α), oncostatin M (OSM) and lipopolysaccharide (LPS). Cathepsin K activity in cartilage explants (control and osteoarthritic-OA) and freshly harvested cartilage (control and OA) was compared to that of MMPs employing C2K77 and C1,2C immunoassays. RESULTS: The addition of Cathepsin K to normal cartilage caused a significant increase (P < 0.01) in the C2K77 epitope release. Whereas the content of C1,2C, that reflects MMP collagenase activity, was increased in media by the addition to cartilage explants of TNF-α and OSM (P < 0.0001) or IL-1β and OSM (P = 0.002), no change was observed in C2K77 which also unchanged in OA cartilages compared to normal. CONCLUSIONS: The ELISA C2K77 measured the activity of cathepsin K in equinecartilage which was unchanged in OA cartilage. Cytokines that upregulate MMP collagenase activity had no effect on endogenous cathepsin K activity, suggesting a different activation mechanism that requires further study.