A sodium dodecyl sulfate–polyacrylamide gel electrophoresis–liquid chromatography tandem mass spectrometry analysis of bovine cartilage tissue response to mechanical compression injury and the inflammatory cytokines tumor necrosis factor α and interleukin‐1β

Abstract

Objective. To compare the response of chondrocytes and cartilage matrix to injurious mechanical compression and treatment with interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣), 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-1␤ or 100 ng/ml of TNF␣ or were subjected to uniaxial, radiallyunconfined 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-1␤ and TNF␣ 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, ␣4-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-1␤ and TNF␣ 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.Osteoarthritis (OA) is characterized by cartilage degeneration, which results from an imbalance between matrix synthesis and matrix degradation. Development of OA secondary to traumatic joint injury occurs in ϳ15-75% of patients over followup periods of 14-22 years, equivalent to an average relative risk or odds ratio of between 3 and 20 of developing OA postinjury (for review, see ref. 1). Moreover, corrective surgery has little or no impact on the risk of developing OA following traumatic joint injury (1). In vitro models of joint injury have helped to understand the contribution of

Structural and Thermodynamic Insights into Chitooligosaccharide Binding to Human Cartilage Chitinase 3-like Protein 2 (CHI3L2 or YKL-39)

“…The fluorescence intensity data for each chitooligosaccharide were fitted reasonably well by the single-site binding model of a nonlinear regression function (Fig. 7B, middle and right top panels for GlcNAc 2 and GlcNAc 3 , respectively, and lower panel from left to right for GlcNAc 4 , GlcNAc 5 , and GlcNAc 6 , respectively). The estimated K d values of YKL-39 WT are summarized in Table 4.…”

“…Human chitinases and chitinase-like proteins (CLPs) 4 are members of the glycosyl hydrolase family 18 (GH-18), involved in several physiological processes such as tissue remodeling, injury, and inflammation (1)(2)(3)(4). Although human chitotriosidase (CHIT1) and acidic mammalian chitinase are active enzymes that hydrolyze natural chitins (5)(6)(7)(8), CLPs are nonenzymatic homologs that possess the chitin-binding groove on the surface of their (␤/␣) 8 TIM barrel domains, which permits these proteins to bind to chitooligosaccharides with high affinity (9 -11).…”

Mechanical Injury and Cytokines Cause Loss of Cartilage Integrity and Upregulate Proteins Associated with Catabolism, Immunity, Inflammation, and Repair

“…5c). Such release of intracellular proteins may result from mechanical disruption of the cells (lysis, necrosis) and/or loss of membrane integrity in cells undergoing apoptosis (16). Although cytokines are unlikely to cause necrosis, TNF-␣ in particular may induce apoptosis.…”

“…5c). This release may be a consequence of acute lysis, necrosis, or cellular leakage following apoptosis (16).…”

A proteomic approach for identification and localization of the pericellular components of chondrocytes

“…The proteomics of the PCM confirmed the dominance of type VI collagen and, moreover, revealed type VI collagen fragmentation in the PCM. Type VI collagen fragments were also found in cartilage explants treated with interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), or mechanical compression (Stevens et al 2008). Disruption of type VI collagen in the PCM seems to destabilize chondrocyte phenotype (Murray et al 2010).…”