1 Endocrinology, Department of Clinical Research, Det Sundhedsvidenskabelige Fakultet, SDU2 KMEB, Department of Clinical Research, Det Sundhedsvidenskabelige Fakultet, SDU3 unknown4 Department of Cancer and Inflammation Research, Department of Molecular Medicine, Det Sundhedsvidenskabelige Fakultet, SDU5 Endocrinology, Department of Clinical Research, Det Sundhedsvidenskabelige Fakultet, SDU6 Department of Cancer and Inflammation Research, Department of Molecular Medicine, Det Sundhedsvidenskabelige Fakultet, SDU
Objective The specific degradation of type II collagen and aggrecan by matrix metalloproteinase (MMP)-9, -13 and ADAMTS-4 and -5 (aggrecanase-1 and -2) in the cartilage matrix is a critical step in pathology of osteoarthritis (OA). The aims of this study were: i) To investigate the relative contribution of ADAMTS-4 and ADAMTS-5 to cartilage degradation upon catabolic stimulation; ii) To investigate the effect of regulating the activities of key enzymes by mean of broad-spectrum inhibitors. Methods Bovine full-depth cartilage explants stimulated with tumor necrosis factor alpha (TNF-α) and Oncostatin M (OSM) were cultured for 21 days with or without a number of inhibitors targeting different types of proteases. Monoclonal antibodies were raised against the active sites of ADAMTS-4, -5, MMP-9 and -13, and 4 ELISAs were developed and technically validated. In addition, the established AGNxI (ADAMTS-degraded aggrecan), AGNxII (MMP-degraded aggrecan), and CTX-II (MMP-derived type II collagen) were quantified in the explants-conditioned media. Results We found that: i) Active ADAMTS-4, MMP-9, -13 were released in the late stage of TNF-α/ OSM stimulation, whereas no significant active ADAMTS-5 was detected in either extracts or supernatants; ii) Active ADAMTS-4 was primarily responsible for E373-374A bond cleavage in aggrecan in this setting; and iii) The compensatory mechanism could be triggered following the blockage of the enzyme caused by inhibitors. Conclusions ADAMTS-4 appeared to be the major protease for the generation of 374ARGS aggrecan fragment in the TNF-α/OSM stimulated bovine cartilage explants. This study addresses the need to determine the roles of ADAMTS-4 and ADAMTS-5 in human articular degradation in OA and hence identify the attractive target for slowing down human cartilage breakdown.
P L O S One, 2015, Vol 10, Issue 4
ADAM Proteins; Aggrecans; Animals; Area Under Curve; Autoantibodies; Biomarkers; Cartilage, Articular; Cattle; Collagen; Disease Models, Animal; Matrix Metalloproteinases; Oncostatin M; Osteoarthritis; Procollagen N-Endopeptidase; Protease Inhibitors; Proteolysis; Tumor Necrosis Factor-alpha