Paediatrics (RCH) - Research Publications

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Author: Fosang, Amanda × Author: Jackson, David × Type: Journal Article ×

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    Bioactivity in an Aggrecan 32-mer Fragment Is Mediated via Toll-like Receptor 2
    Lees, S ; Golub, SB ; Last, K ; Zeng, W ; Jackson, DC ; Sutton, P ; Fosang, AJ (WILEY, 2015-05)
    OBJECTIVE: To determine whether an aggrecan 32-mer fragment derived from dual ADAMTS and matrix metalloproteinase (MMP) cleavage in the aggrecan interglobular domain was bioactive and, if so, to elucidate its mechanism of action. METHODS: Mouse primary chondrocytes, synovial fibroblasts, or peritoneal macrophages, human primary chondrocytes, and cells or cell lines from myeloid differentiation factor 88 (MyD88)-deficient and Toll-like receptor 2 (TLR-2)-deficient mice were stimulated with synthetic mouse 32-mer peptide, human 32-mer peptide, a 32-mer scrambled peptide, or native, glycosylated 32-mer peptide. Cells stimulated with 32-mer peptide were analyzed for changes in messenger RNA (mRNA) expression by quantitative polymerase chain reaction. Conditioned medium was analyzed for levels of interleukin-6 protein by an AlphaLISA or for levels of MMP-3 and MMP-13 protein by Western blotting. NF-κB activation was measured in a luciferase reporter assay. RESULTS: Treatment of mouse cells or cartilage explants with 32-mer peptide or scrambled peptide revealed that the 32-mer peptide, but not the scrambled peptide, had antianabolic, procatabolic, and proinflammatory bioactivity in vitro. Chondrocytes, synovial fibroblasts, and macrophages from MyD88-deficient mice failed to respond to 32-mer peptide stimulation. A macrophage cell line derived from TLR-2-deficient mice also failed to respond to 32-mer peptide stimulation. Stimulation of human chondrocytes with human 32-mer peptide increased the expression of catabolic markers at the mRNA and protein levels. Mouse and human 32-mer peptide stimulated NF-κB activation in a TLR-2-dependent reporter assay, and the response of chondrocytes from both species to native, glycosylated 32-mer peptide was similar to the response to synthetic peptides. CONCLUSION: The aggrecan 32-mer fragment is a novel endogenous ligand of TLR-2 with the potential to accelerate cartilage destruction in vivo.
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    Antibodies to MMP-cleaved aggrecan.
    Fosang, AJ ; Last, K ; Jackson, DC ; Brown, L (Humana Press, 2001)