Author(s): Stevens AL, Wishnok JS, Chai DH, Grodzinsky AJ, Tannenbaum SR
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Abstract OBJECTIVE: To compare the response of chondrocytes and cartilage matrix to injurious mechanical compression and treatment with interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), by characterizing proteins lost to the medium from cartilage explant culture. METHODS: Cartilage explants from young bovine stifle joints were treated with 10 ng/ml of IL-1beta or 100 ng/ml of TNFalpha or were subjected to uniaxial, radially-unconfined injurious compression (50\% strain; 100\%/second strain rate) and were then cultured for 5 days. Pooled media were subjected to gel-based separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and analysis by liquid chromatography tandem mass spectrometry, and the data were analyzed by Spectrum Mill proteomics software, focusing on protein identification, expression levels, and matrix protein proteolysis. RESULTS: More than 250 proteins were detected, including extracellular matrix (ECM) structural proteins, pericellular matrix proteins important in cell-cell interactions, and novel cartilage proteins CD109, platelet-derived growth factor receptor-like, angiopoietin-like 7, and adipocyte enhancer binding protein 1. IL-1beta and TNFalpha caused increased release of chitinase 3-like protein 1 (CHI3L1), CHI3L2, complement factor B, matrix metalloproteinase 3, ECM-1, haptoglobin, serum amyloid A3, and clusterin. Injurious compression caused the release of intracellular proteins, including Grp58, Grp78, alpha4-actinin, pyruvate kinase, and vimentin. Injurious compression also caused increased release and evidence of proteolysis of type VI collagen subunits, cartilage oligomeric matrix protein, and fibronectin. CONCLUSION: Overload compression injury caused a loss of cartilage integrity, including matrix damage and cell membrane disruption, which likely occurred through strain-induced mechanical disruption of cells and matrix. IL-1beta and TNFalpha caused the release of proteins associated with an innate immune and stress response by the chondrocytes, which may play a role in host defense against pathogens or may protect cells against stress-induced damage.
This article was published in Arthritis Rheum
and referenced in Journal of Neurological Disorders