J M Milner1, S F Elliott, T E Cawston. 1. Department of Rheumatology, The Medical School, University of Newcastle-upon-Tyne, England, UK.
Abstract
OBJECTIVE: Bovine and human cartilages in explant culture respond to proinflammatory cytokines with the up-regulation of procollagenases. In stimulated bovine nasal cartilage (BNC), >90% of collagen is released by day 14 of culture, but collagen release is rarely seen before day 7. The aim of this study was to investigate if activation of procollagenases is a rate-limiting step in cartilage collagen breakdown. METHODS: BNC and human articular cartilage explants were cultured with interleukin-1alpha (IL-1alpha) and/or oncostatin M (OSM) with or without test reagents. Collagen levels were determined by assay of hydroxyproline. Collagenase activity was measured using the diffuse fibril assay. RESULTS: The addition of procollagenase activators, matrix metalloproteinase 3 (MMP-3), and APMA to IL-1alpha/OSM-stimulated BNC resulted in early release of collagen. The release with APMA was completely blocked by the addition of tissue inhibitor of metalloproteinases 1. This shows that procollagenases are present early in the culture period, but cartilage collagen breakdown does not happen until activation occurs. The addition of plasminogen to IL-1alpha/OSM-stimulated cartilage produced early collagen release in bovine and a significant increase in human cartilage. Thus, plasminogen activators (PAs) are present and convert plasminogen to plasmin, a known activator of several MMPs, including collagenases. Addition of alpha1-proteinase inhibitor or a urokinase-type PA inhibitor, 7-amino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, partially blocked the breakdown of collagen from IL-1alpha/OSM-treated bovine cartilage. This suggests that serine proteinases are involved in the activation cascades of procollagenases that result in cartilage collagen breakdown. CONCLUSION: The activation of procollagenases is a key control point in cartilage collagen breakdown, and serine proteinase pathways activate MMPs.
OBJECTIVE:Bovine and human cartilages in explant culture respond to proinflammatory cytokines with the up-regulation of procollagenases. In stimulated bovinenasal cartilage (BNC), >90% of collagen is released by day 14 of culture, but collagen release is rarely seen before day 7. The aim of this study was to investigate if activation of procollagenases is a rate-limiting step in cartilage collagen breakdown. METHODS: BNC and humanarticular cartilage explants were cultured with interleukin-1alpha (IL-1alpha) and/or oncostatin M (OSM) with or without test reagents. Collagen levels were determined by assay of hydroxyproline. Collagenase activity was measured using the diffuse fibril assay. RESULTS: The addition of procollagenase activators, matrix metalloproteinase 3 (MMP-3), and APMA to IL-1alpha/OSM-stimulated BNC resulted in early release of collagen. The release with APMA was completely blocked by the addition of tissue inhibitor of metalloproteinases 1. This shows that procollagenases are present early in the culture period, but cartilage collagen breakdown does not happen until activation occurs. The addition of plasminogen to IL-1alpha/OSM-stimulated cartilage produced early collagen release in bovine and a significant increase in human cartilage. Thus, plasminogen activators (PAs) are present and convert plasminogen to plasmin, a known activator of several MMPs, including collagenases. Addition of alpha1-proteinase inhibitor or a urokinase-type PA inhibitor, 7-amino-4-chloro-3-(3-isothiureidopropoxy) isocoumarin, partially blocked the breakdown of collagen from IL-1alpha/OSM-treated bovine cartilage. This suggests that serine proteinases are involved in the activation cascades of procollagenases that result in cartilage collagen breakdown. CONCLUSION: The activation of procollagenases is a key control point in cartilage collagen breakdown, and serine proteinase pathways activate MMPs.
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