Tenascin-C fragments are endogenous inducers of cartilage matrix degradation
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Cartilage destruction is a hallmark of osteoarthritis (OA) and is characterized by increased protease activity resulting in the degradation of critical extracellular matrix (ECM) proteins essential for maintaining cartilage integrity. Tenascin-C (TN-C) is an ECM glycoprotein, and its expression is upregulated in OA cartilage. We aimed to investigate the presence of TN-C fragments in arthritic cartilage and establish whether they promote cartilage degradation. Expression of TN-C and its fragments was evaluated in cartilage from subjects undergoing joint replacement surgery for OA and RA compared with normal subjects by western blotting. The localization of TN-C in arthritic cartilage was also established by immunohistochemistry. Recombinant TN-C fragments were then tested to evaluate which regions of TN-C are responsible for cartilage-degrading activity in an ex vivo cartilage explant assay measuring glycosaminoglycan (GAG) release, aggrecanase and matrix metalloproteinase (MMP) activity. We found that specific TN-C fragments are highly upregulated in arthritic cartilage. Recombinant TN-C fragments containing the same regions as those identified from OA cartilage mediate cartilage degradation by the induction of aggrecanase activity. TN-C fragments mapping to the EGF-L and FN type III domains 3–8 of TN-C had the highest levels of aggrecan-degrading ability that was not observed either with full-length TN-C or with other domains of TN-C. TN-C fragments represent a novel mechanism for cartilage degradation in arthritis and may present new therapeutic targets for the inhibition of cartilage degradation.
KeywordsTenascin-C Extracellular matrix Damage-associated molecular patterns Aggrecanases Matrix metalloproteinases
A disintegrin and metalloproteinase with Thrombospondin-like motifs
Epidermal growth factor
Hepatocyte growth factor
Tumour necrosis factor
Osteoarthritis (OA) is the most common joint disorder affecting increasing numbers of our ageing populations [1, 2]. Factors contributing to tissue damage include cytokine release, superoxide production and protease activation ultimately leading to loss of joint function [3, 4, 5, 6]. Current treatments are largely focused around control of pain and maintenance of function, since there are no effective disease-modifying treatments for OA . There is therefore an unmet need to gain a deeper understanding of the mechanisms underlying joint destruction in OA.
A number of factors influence ongoing cartilage damage in OA, including injurious mechanical compression of cartilage that alters gene transcription of degradative enzymes including MMPs and aggrecanases (ADAMTSs) [8, 9]. Such proteases degrade the key extracellular matrix (ECM) components of cartilage including type II collagen and aggrecan [10, 11]. Several studies have shown increased production of ECM molecules during the early stages of OA [12, 13, 14]. Type II collagen, cartilage oligomeric matrix protein (COMP), fibronectin, fibromodulin and tenascin-C are among the ECM proteins that are altered in OA cartilage both at the messenger RNA and protein level. As ongoing protease-mediated cartilage destruction ensues, fragments of ECM proteins are generated, which accumulate during ongoing disease progression.
Recent reports suggest that specific ECM proteins become endogenous catabolic factors potentiating further joint damage in OA. Activation of pro-inflammatory pathways by such ECM proteins has led to their description as damage-associated molecular patterns (DAMPs). Several groups have shown that fragments of ECM proteins acquire novel proteolytic activity upon fragmentation that was hitherto absent in the full-length molecule. For example, a fragment of type II collagen localizing to the N-terminus of the molecule upregulates mRNA and protein levels of MMP-2, MMP-3, MMP-9 and MMP-13 in bovine chondrocytes and explants . Other ECM molecules showing distinct activity upon fragmentation include fibromodulin, hyaluronan and fibronectin [16, 17, 18]. However, it is unclear whether these are the only protein fragments responsible for further cartilage destruction. Tenascin-C (TN-C) is an ECM glycoprotein associated with tissue injury and repair. TN-C is a hexameric protein of 1.5 million Da comprising an assembly domain, epidermal growth factor-like repeats (EGF-L), fibronectin type III repeats (TNIII) and a fibrinogen-like globe (FBG) . Little TN-C is expressed in normal human joints, but expression is increased in cartilage [20, 21], synovial tissue [22, 23] and synovial fluid  in OA and RA. TN-C potentiates chronic inflammation in RA by the upregulation of pro-inflammatory cytokines, and this effect is not observed in TN-C knockout mice . In contrast, in a murine model of OA, TN-C knockout mice showed evidence of delayed healing and repair . The increased levels of TN-C reported in OA tissue and the homology of TN-C domains to other known DAMPs in cartilage prompted us to examine whether TN-C fragments exist in arthritic cartilage and whether TN-C fragments possess novel cartilage-degrading ability.
Materials and methods
Preparation of human cartilage extracts
Human cartilage samples from patients undergoing joint replacement surgery for OA of the knee/hip or normal cartilage obtained from patients undergoing amputations for trauma were obtained with full Ethics Approval (REC reference number: 09/H0806/45) from St George’s and Heatherwood Hospitals NHS Trust. All participants donated tissue with full informed consent. All reagents were obtained from Sigma-Aldrich unless otherwise stated. Human cartilage was washed three times in sterile PBS, and a total of 0.1 g was collected per subject. The cartilage was further dissected in 1 ml of 2 × reducing sample buffer containing 0.2 g SDS, 5 ml upper gel buffer consisting of 84 mM ammediol, 62 mM HCl and 0.02% sodium azide, 1 ml bromophenol blue, 4 ml glycerol and 880 μl of 0.5 M EDTA (pH 8.0) to a final volume of 11 ml. The dissected cartilage and sample buffer were carefully transferred to a 1.5-ml Eppendorf tube and boiled at 100°C for 10 min. The samples were stored at −20°C until further use.
Immunohistochemistry of human cartilage
Fresh cartilage for sectioning was obtained at the time of joint surgery from subjects and immediately fixed in 4% paraformaldehyde in 0.1% phosphate-buffered saline (PBS; pH 7.4) and then sectioned using a cryostat into 7 micron slices onto polylysine-coated glass slides (VWR, Leuven, Belgium). To block endogenous peroxidase activity, 3% H2O2 was applied in the dark for 15 min. Sections were then blocked with 2% goat serum for 30 min. Following this, sections were incubated with an anti-TN-C monoclonal antibody raised against the N-terminal heptad region of TN-C (MAB 1908, Millipore, Watford, UK) at 1:1,000 for 1 h at room temperature, followed by incubation with Dako REAL EnVision detection system/HRP for rabbit/mouse secondary antibodies for 30 min (Dako, Denmark). For control experiments, a mouse IgG primary antibody (SC-2025) (Santa Cruz, Heidelburg, Germany) was used. Signal was developed with the Dako REAL DAB + chromogen substrate system according to the manufacturers’ instructions. Slides were counterstained with haematoxylin and mounted with Histomount (National Diagnostics, Atlanta, USA). All slides were viewed with a Zeiss AXIOPLAN 2 light microscope running on Axiovision system 4.7.
SDS–PAGE and western blotting
Proteins were resolved by SDS/PAGE with reduction using the ammediol/glycine/HCl buffer system of Wyckoff et al.  for the Tris–glycine buffer method according to Laemmli . Gels were typically run for 60 min at 200 V.
Staining with Coomassie Brilliant Blue R-250 and silver was performed as previously described [29, 30]. For western blotting, gels were electrotransferred onto a PVDF membrane at 25 V (constant voltage) for 240 min in 200 ml transfer buffer containing 20% v/v methanol, 7.2 g glycine and 1.512 g Tris base. Five percentage BSA was used for blocking, for 30 min following which the primary anti-TN-C antibody in 1% BSA/TBS for 1 h (dilution 1:1,000). The membrane was washed three times with TBS/0.05% Tween. After washing, a 1:5,000 dilution of an AP-linked secondary antibody (Promega, Southampton, UK) in 1% BSA/TBS was added and incubated for 1 h. Membranes were washed three times in TBS/0.05% Tween and incubated with AP substrate for development for up to 30 min.
Tenascin-C and recombinant TN-C proteins
Full-length tenascin-C (Millipore, Watford, UK) was dialysed into TBS buffer (50 mM Tris–HCl, 150 mM NaCl, pH 7.5) before use in cartilage explant cultures. Recombinant TN-C domains were produced as previously described . The Limulus amoebocyte lysate (LAL) assay (Cambrex, Wiesbaden, Germany) was used to test the amount of LPS in all purified and recombinant TN-C proteins as previously described . A standard curve was performed for each experiment using E.coli-derived LPS that was provided in the kit. Detection levels for purified proteins were undetectable in the 1–100 pg/ml range, which is well below the levels required to stimulate cartilage degradation.
Measurement of glycosaminoglycan release, aggrecanase and MMP activity
Fresh porcine metacarpophalangeal joints of 3- to 9-month-old pigs (Turners’, Hampshire, UK) or human tissue were obtained with full Ethical Approval (REC reference number: 09/H0806/45) and dissected into pieces 3 mm × 2–3 mm wide (about 10 mg) as we have previously described [31, 33]. After dissection, cartilage was rested for 48 h at 37°C under 5% CO2 in DMEM containing 5% foetal calf serum, penicillin/streptomycin (100 units/ml), amphotericin B (100 units/ml) and 10 mM HEPES. Each cartilage piece was placed in one well of a round bottom 96-well plate with 200 μl of serum-free medium with full-length TN-C, recombinant TN-C fragments, IL-1α (a gift from Prof J Saklatvala, Imperial College London) as a positive control. Each treatment was performed in triplicate. After 48 h, media and cartilage were harvested and stored at −20°C until use. Each experiment was performed at least 3 times on separate porcine trotters.
The total GAG released into media by cartilage was measured using a modification of the DMMB assay . A volume of 250 μl of the DMMB assay reagent (16 mg/ml DMMB, 41 mM sodium chloride, 40 mM glycine, 9.5 mM HCl) was added to 2.5 μl of culture medium in a 96-well plate, and then the absorbance was read immediately at 540 nm. Each sample was assayed in duplicate. The optical density from the experimental samples was converted into μg of chondroitin from the standard curve.
Aggrecanase and MMP activity in the conditioned medium of treated cartilage was determined using neoepitope assays as previously described [31, 33]. Aggrecanase and MMP-specific neoepitope antibodies anti-ARGSV (BC-3) and anti-FFGVG (BC-14), respectively, were kindly provided by Professor Bruce Caterson and Dr Clare Hughes (University of Wales, Cardiff).
The GAG release assay in cartilage explants was evaluated using Graphpad Prism software (San Diego, California). Mean values were calculated for each treatment in triplicate and expressed as mean ± standard error of mean. Significance was analysed with two-tailed Student’s t tests and defined as P < 0.05.
Full-length TN-C and TN-C fragments are upregulated in arthritic cartilage
Recombinant TN-C fragments induce glycosaminoglycan release and aggrecanase activity in cartilage
In this study, we have shown that two main forms of fragmented TN-C of 100 kDa and 150 kDa are detectable in arthritic cartilage but not in normal subjects. The TN-C fragments we have detected map to the EGF-L and FN type III 3–8 domains in arthritic human cartilage. Recombinant TN-C fragments comprising the same domains induce GAG release and aggrecanase activity in cartilage in a dose-dependent manner. Although increased TN-C expression has previously been described in arthritic cartilage [20, 21, 34], our study is the first to describe and characterize TN-C fragments in cartilage. Zhen et al.  recently showed that human cartilage subjected to digestion with ADAMTS-5 cleaves TN-C peptides that map to the N-terminal TA domain when digested cartilage was analysed by mass spectrometry. It is therefore plausible that the generation of TN-C fragments by proteases such as ADAMTS-5 generates TN-C fragments which then stimulate a positive feedback loop of further cartilage matrix degradation mediated by protease activation including aggrecanases as found in our study.
Work conducted in other tissues has shown that leg ulcer exudates have high levels of TN-C fragments  and the EGF-L repeats are increased in blood vessels of subjects undergoing carotid endarterectomy . Specific TN-C fragments have distinct functions: the EGF-L domains influence neuronal migration in development [38, 39] and induce apoptosis in smooth muscle cells . The FN III domains of TN-C are susceptible to proteolytic cleavage  and interact with ligands including integrins , heparin binding  and binding to other ECM molecules including fibronectin . Whereas previous work has shown that full-length TN-C is involved in wound repair in OA [26, 34], our work has shown that when TN-C is cleaved to generate fragmented forms, such TN-C fragments acquire novel proteolytic activity that mediates catabolic pathways in articular cartilage. There are therefore differences between the full-length and fragmented forms of TN-C, which mediate anabolic and catabolic pathways, respectively, in OA models.
This work was funded by a Clinical Research Training Fellowship from The Wellcome Trust to Dr Nidhi Sofat, grant code 070848 and an Enterprise Award from St George’s, University of London. We thank Professor Bruce Caterson and Dr Clare Hughes (Cardiff University) for BC-3 and BC-14 antibodies. We thank Dr Kim Midwood (Imperial College London) for recombinant tenascin-C proteins and Professor Hideaki Nagase for useful discussions. We thank Miss Kinga Anita Szewczyk, Mr Ray Moss and Miss Kay Elderfield for assistance with immunohistochemistry (St George’s, University of London).
Conflict of interest
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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