Paediatrics (RCH) - Research Publications
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ItemBioactivity in an Aggrecan 32-mer Fragment Is Mediated via Toll-like Receptor 2(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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ItemNo Preview AvailableCytokine-Induced Increases in ADAMTS-4 Messenger RNA Expression Do Not Lead to Increased Aggrecanase Activity in ADAMTS-5-Deficient Mice(WILEY, 2010-11)OBJECTIVE: To compare the regulation of aggrecanase messenger RNA (mRNA) and enzyme activity by proinflammatory cytokines in primary mouse chondrocytes. METHODS: Primary chondrocytes were isolated from knee epiphyses of 6-8-day-old mice and cultured as monolayers. The cells were incubated with tumor necrosis factor α (TNFα), oncostatin M (OSM), or interleukin-6 (IL-6)/soluble IL-6 receptor, and mRNA levels were measured by quantitative polymerase chain reaction at various time points. To measure aggrecanase activity, the cells were incubated with cytokine in the presence of exogenous aggrecan, and substrate cleavage was measured using antibodies to neoepitopes. RESULTS: Expression of both ADAMTS-4 and ADAMTS-5 mRNA was up-regulated by TNFα and OSM. ADAMTS-5 mRNA expression was also up-regulated by IL-6. Treatment of wild-type mouse chondrocytes with each of the 3 cytokines increased cleavage of aggrecan at Glu(373)↓(374) Ala and Glu(1670)↓(1671) Gly; in chondrocytes lacking ADAMTS-5 activity, there was negligible cleavage at either site despite increased expression of ADAMTS-4 mRNA in the presence of TNFα or OSM. None of the cytokines substantially altered mRNA expression of ADAMTS-1 or ADAMTS-9. CONCLUSION: Despite substantial increases in the expression of ADAMTS-4 mRNA induced by TNFα and OSM, these cytokines induced little if any increase in aggrecanolysis in ADAMTS-5-deficient mouse chondrocytes. Our data show a poor correlation between the level of cytokine-induced ADAMTS-4 mRNA expression and the level of aggrecan-degrading activity in cultured chondrocytes.
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ItemNo Preview AvailableBIOACTIVITY IN AN AGGRECAN 32MER FRAGMENT IS MEDIATED VIA TOLL-LIKE RECEPTORS(ELSEVIER SCI LTD, 2014-04)
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ItemEvidence for lysosomal exocytosis and release of aggrecan-degrading hydrolases from hypertrophic chondrocytes, in vitro and in vivo(COMPANY OF BIOLOGISTS LTD, 2012-04-15)The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca(2+)-dependent lysosomal exocytosis. In this study we confirm that hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development. We show that lysosome-associated membrane protein 1 (LAMP1) is detected at the cell surface following in vitro treatment of epiphyseal chondrocytes with the calcium ionophore, ionomycin. Furthermore, we show that in addition to the lysosomal exocytosis markers, cathepsin D and β-hexosaminidase, ionomycin induces release of aggrecan- and hyaluronan-degrading activity from cultured epiphyseal chondrocytes. We identify VAMP-8 and VAMP7 as v-SNARE proteins with potential roles in lysosomal exocytosis in hypertrophic chondrocytes, based on their colocalisation with LAMP1 at the cell surface in secondary ossification centers in mouse tibiae. We propose that resorbing growth plate cartilage involves release of destructive hydrolases from hypertrophic chondrocytes, via lysosomal exocytosis.
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ItemInvestigating ADAMTS-mediated aggrecanolysis in mouse cartilage(NATURE PUBLISHING GROUP, 2011-03)Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix. The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7-11 d to complete.
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ItemNo Preview AvailableA Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2016-02-12)The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.
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ItemAggrecanase Cleavage in Juvenile Idiopathic Arthritis Patients Is Minimally Detected in the Aggrecan Interglobular Domain but Robust at the Aggrecan C-Terminus(WILEY-BLACKWELL, 2012-12)OBJECTIVE: To investigate aggrecan degradation in juvenile idiopathic arthritis (JIA). METHODS: The pattern and abundance of aggrecan fragments in synovial fluid (SF) aspirates from JIA patients were analyzed and compared with aggrecan fragments in SF from patients with other arthritides, children with knee injury, and a knee-healthy reference group. Concentrations of sulfated glycosaminoglycan (sGAG) in SF were measured by Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density-gradient centrifugation, deglycosylated, and analyzed by Western blot using antibodies specific for either aggrecanase-derived ARGS, SELE, and KEEE neoepitopes or the aggrecan G3 domain. RESULTS: The concentration of sGAG in SF from patients with JIA was significantly lower compared with that in SF from patients with osteoarthritis (OA) (P < 0.001), patients with juvenile knee injury (P = 0.006), and knee-healthy controls (P = 0.022). Western blot analysis revealed KEEE, SELE, and G3 fragments generated by aggrecanase cleavage in the chondroitin sulfate-rich region of aggrecan in patients with JIA. The pattern of aggrecan fragments in JIA patients was not identical to that in pooled OA SF, although there were notable similarities. Surprisingly, aggrecanase-derived ARGS fragments were barely detectable in JIA SF, in marked contrast to levels in OA SF. CONCLUSION: Aggrecanases appear to cleave minimally in the interglobular domain of aggrecan in JIA patients despite robust levels of cleavage in the chondroitin sulfate-rich region. These results suggest that in JIA, unlike other arthritides, aggrecanase cleavage in the aggrecan interglobular domain might not be a major pathogenic event.