- Email: [email protected]
C2K77 ELISA detects cleavage of type II collagen by cathepsin K in equine articular cartilage
Accepted Manuscript C2K77 ELISA detects cleavage of type II collagen by cathepsin K in equine articular cartilage Beatriz Noé, A. Robin Poole, John S. Mort, Helene Richard, Guy Beauchamp, Sheila Laverty PII:
S1063-4584(17)31159-7
DOI:
10.1016/j.joca.2017.08.011
Reference:
YJOCA 4074
To appear in:
Osteoarthritis and Cartilage
Received Date: 20 January 2017 Revised Date:
21 August 2017
Accepted Date: 26 August 2017
Please cite this article as: Noé B, Poole AR, Mort JS, Richard H, Beauchamp G, Laverty S, C2K77 ELISA detects cleavage of type II collagen by cathepsin K in equine articular cartilage, Osteoarthritis and Cartilage (2017), doi: 10.1016/j.joca.2017.08.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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C2K77 ELISA detects cleavage of type II collagen by cathepsin K in equine articular cartilage
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Authors: Beatriz Noé^*, A. Robin Pooleǂ, John S. Mortǂ, Helene Richard^, Guy Beauchamp¶, Sheila
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Laverty^
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Affiliation : ^Comparative Orthopaedic Research Laboratory, Département de sciences cliniques,
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Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe (QC) J2S
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2M2, Canada
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ǂDivision of Orthopaedics, Department of Surgery, McGill University, Montreal, (QC), Canada
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¶ Département de Pathologie et Microbiologie Vétérinaires, Faculté de Médecine Vétérinaire,
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Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe (QC) J2S 2M2, Canada
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*Beatriz Noé’s current address is : Département de sciences cliniques, Faculté de Médecine Vétérinaire,
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Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe (QC) J2S 2M2, Canada. Tel 1-438-881-2892.
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E-mail : [email protected]
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Corresponding author: S Laverty MVB, DACVS & DECVS, Département de sciences cliniques, Faculté
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de Médecine Vétérinaire, Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe (QC) J2S 2M2,
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Canada. Tel1-450 7788100. Email: [email protected]
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Abstract Objectives: Develop a species-specific ELISA for a neo-epitope generated by cathepsin K
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cleavage of equine type II collagen to: 1) measure cartilage type II collagen degradation by cathepsin K
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in vitro, 2) identify cytokines that upregulate cathepsin K expression and 3) compare cathepsin K with
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MMP collagenase activity in stimulated cartilage explants and freshly isolated normal and OA articular
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cartilages.
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Design: A new ELISA (C2K77) was developed and tested by measuring the activity of
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exogenous cathepsin K on equine articular cartilage explants. The ELISA was then employed to
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measure endogenous cathepsin K activity in cultured cartilage explants with or without stimulation by
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interleukin-1 beta (IL-1β), tumour necrosis-alpha (TNF-α), oncostatin M (OSM) and lipopolysaccharide
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(LPS). Cathepsin K activity in cartilage explants (control and osteoarthritic-OA) and freshly harvested
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cartilage (control and OA) was compared to that of MMPs employing C2K77 and C1,2C immunoassays.
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Results: The addition of Cathepsin K to normal cartilage caused a significant increase (p˂0.01) in
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the C2K77 epitope release. Whereas the content of C1,2C, that reflects MMP collagenase activity, was
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increased in media by the addition to cartilage explants of TNF-α and OSM (p˂0.0001) or IL-1β and
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OSM (p = 0.002), no change was observed in C2K77 which also unchanged in OA cartilages compared
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to normal.
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Conclusions: The ELISA C2K77 measured the activity of cathepsin K in equine cartilage which was
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unchanged in OA cartilage. Cytokines that upregulate MMP collagenase activity had no effect on
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endogenous cathepsin K activity, suggesting a different activation mechanism that requires further
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study.
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Keywords
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cathepsin K, type II collagen, ELISA, osteoarthritis, cartilage, MMPs
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Introduction Osteoarthritis (OA) is a progressive degenerative joint disease characterized by articular cartilage
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degeneration, subchondral bone remodelling, synovial inflammation and pain1. Many factors can trigger
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OA including joint trauma and overuse, aging, obesity2 and genetic predisposition3.
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Type II collagen, the most abundant collagen in the extracellular matrix of articular cartilage,
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represents 95% of the total collagen content4. The collagen fibril is composed of a triple helix of three
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identical α-chains, imparting tensile strength essential for maintaining the integrity of cartilage4. In OA,
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this collagen is progressively degraded by enzymatic digestion, essentially an irreversible event in
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cartilage degeneration in OA5.
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Few vertebrate proteases are able to cleave intact type II collagen. Following primary cleavage,
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and molecule unwinding, it becomes susceptible to additional cleavage by many extracellular proteases.
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Thus, primary cleavage is a key step in cartilage matrix degradation in OA6.
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Matrix metalloproteinase collagenases (MMPs) are involved in this primary cleavage in OA6.
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MMPs 1, 8 and 13 can cleave type II collagen near the C-terminal end: MMP 13 may be especially
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responsible in OA7. But cathepsin K, may also play an important role in cartilage degradation in OA. Cathepsin K can cleave intact type II collagen at multiple sites
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. It is a cysteine protease
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capable of degrading many cartilage matrix molecules including proteoglycans10, in the case if aggrecan
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at several sites in the G1, interglobular and chondroitin sulfate domains10. Originally found in osteoclasts,
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playing an important role in bone resorption11, it is also present in chondrocytes12. In contrast to the
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MMPs, cathepsin K cleaves type II collagen close to the N-terminus at various helical sites8. Our studies
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have revealed an increased abundance of cathepsin K, and its degradation products of cleaved type II
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collagen, adjacent to chondrocytes in OA cartilage suggesting it plays a role in matrix degradation in
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equine12 and human5, 13 joint disease. The capacity of cytokines and other molecules to stimulate MMP collagenase activity involved
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in type II collagen degradation in OA has been well studied14-16 but little is known about the regulation
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of cathepsin K. Such knowledge would help us understand its role in disease, including OA, and could
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potentially lead to the development of a biomarker of cartilage degradation in OA.
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Cathepsin K cleavage sites, have been recently identified (Fig. 1). An antibody against one such
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cleavage site (C2K77) has been raised by us in rabbits8. Immunostaining of OA cartilage sections
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revealed the antibody’s capacity to detect cathepsin K cleavage in OA cartilage8 suggesting that the
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antibody may be a suitable candidate for an ELISA immunoassay.
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We hypothesized that cathepsin K activity in cartilage is measurable by a cathepsin K specific
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cleavage neoepitope ELISA, similar to those developed for MMP collagenases14, 17. Our first objective
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was to develop a novel, competitive ELISA inhibition-assay directed against a neoepitope generated by
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the cathepsin K cleavage of equine type II collagen. Our second objective was to measure type II
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collagen degradation by cathepsin K in vitro in normal, stimulated and OA articular cartilages and
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determine whether cytokines regulate cathepsin K activity. Because it may be comparable to that of
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MMP collagenases, our third objective was to compare cathepsin K and collagenase activities using
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specific anti-neoepitope based ELISA assays in stimulated cartilage explants and also in freshly isolated
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control and OA cartilage.
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Methods
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Development of a competitive C2K77 ELISA
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C2K77 ELISA:
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The anti-neoepitope (C2K77) antibody was produced by immunizing rabbits with a synthetic
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peptide, KPGKSGGC, that includes the equine type II collagen cleavage (the most N-terminal cleavage)
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neoepitope generated by cathepsin K8. It has been shown previously8 that this antibody does not
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recognize intact alpha chains of type II collagen and does not react with uncleaved collagen. The C2K77
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immunizing peptide and the purified antibody were titrated to attain the optimal operational
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concentrations for establishment of the competitive C2K77. Sensitivity, reproducibility and recovery
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were assessed. The antibody was tested to detect specificity to equine peptide by immunoassaying 50
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ng/ml of the human C2K77 immunizing peptide. The C2K77 ELISA was then employed to detect the
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specific cathepsin K generated neoepitopes contained in type II collagen cleavage fragments, and
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released from cartilage or present in the cartilage matrix, under a variety of experimental conditions, to
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better understand the involvement of cathepsin K activity in health and pathology.
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Equine articular cartilage
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Articular cartilage was harvested from equine healthy and OA metacarpophalangeal (MCP)
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joints obtained from a local abattoir or from horses donated to the veterinary teaching hospital at the
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Université de Montréal (CHUV). For more details see Supplementary Material.
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Assessment of C2K77 ELISA by digestion of healthy articular cartilage with cathepsin K to
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generate type II collagen cleavage fragment release over time
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Healthy equine cartilages (specimens 1-4; Table 1) were chopped into small pieces and placed in
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Eppendorf tubes containing PBS. Cartilage was then incubated in 2 ml of digestion buffer at 32°C and
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agitated for 24 h with 250 nM recombinant human cathepsin K18. Aliquots (50 µl) of the digestion
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buffer were harvested at 0, 8 and 24 h. Trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-
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64) was added to each aliquot to inactivate cathepsin K. Control cartilage was similarly treated but
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without the addition of cathepsin K. Supernatants in Eppendorf tubes were stored at –20°C until
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immunoassay with the C2K77 ELISA. On termination of the cathepsin K and control digestions, the
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cartilage was collected, blotted dry, weighed and stored at -20°C. In order to solubilize the C2K77
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neoepitope in the cartilage matrix, as in previous studies5, a part of the remaining cartilage was digested
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with α-chymotrypsin. The C2K77 immunizing peptide was mixed with chymotrypsin – 1mg/ml ( BSA
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1mg/ml) for 24 hrs and then assayed to confirm that the enzyme did not digest the peptide. Alpha
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chymotrypsin was then inactivated by the addition of N-tosyl-L-phenylalanine-chloromethyl ketone
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(TPCK). The digestion buffer was analyzed in duplicate with the C2K77 ELISA.
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Demonstration that cathepsin K can cleave type II collagen in situ As part of the study it was important to determine whether we could demonstrate cleavage of
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type II collagen in situ by cathepsin K. In order to address this we digested cartilage in equine
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osteochondral sections containing normal and osteoarthritic cartilage (specimen 32; Table 1). For more
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details see Supplementary Material.
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Ability of cytokines (IL-1β, IL-1β+OSM, TNF-α+OSM) or LPS to stimulate cleavage by cathepsin
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K in cultured healthy equine cartilage explants and comparison to endogenous non-stimulated
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cathepsin K mediated type II collagen cleavage in OA explants
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Full depth articular cartilage was harvested under aseptic conditions from healthy equine MCP
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joints (n = 3) (specimens 6-8, Table 1) and from joints with OA (n=3) (specimens 9-11, Table 1). The
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cartilage was placed in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen Canada Inc.)
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containing antibiotics/anti-mycotic agents (Penicillin 385.6 units/ml, streptomycin 385.6 µg/ml and
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Amphotericin 0.963 µg/ml) for 30 mins at 37°. Two additional washing steps, 15 min per washing, were
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performed with 30 ml of DMEM containing antibiotics/anti-mycotic. The cartilage was then chopped
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into approximately 2 x 3 mm explants and again washed. The cartilage was weighed and 50-70 mg was
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randomly placed into each well of a 24-well tissue culture plates containing 1 ml of DMEM with 10%
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fetal bovine serum (FBS). The cartilage explants were pre-cultured for 2 days at 37°C in a humidified
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incubator with 5% CO2/95% air. The explants were cultured in 24 well plates and four wells were used
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per stimulation, whenever sufficient tissue was available.
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To simulate inflammatory stimuli encountered in joint disease, culture wells contained IL-1β (10
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ng/ml) (Kingfisher Biotech USA) or IL-1β and OSM (1 ng/ml and 10 ng/ml, respectively) (OSM:
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Prepotech, USA) or TNF-α and OSM (10 ng/ml and 10 ng/ml, respectively) (TNF: Preprotech, USA) or
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LPS (10 µg/ml) (Sigma-Aldrich, USA). Wells without these additives served as controls. The culture
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media were harvested and replaced every 7 days, up to day 21, stored in Eppendorf tubes and frozen at -
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20°C.
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Media were immunoassayed employing the C1,2C ELISA (IBEX Technologies Inc., Montreal,
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Quebec, Canada), an immunoassay specific for a neoepitope generated by type II collagen cleavage by
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MMP collagenases, and the C2K77 immunoassay.
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Concentrations of C2K77 and C1,2C neoepitopes in fresh normal and OA cartilage
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Cartilage was harvested from healthy equine joints (n=8; specimens 12-19, Table 1) and joints
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with macroscopic evidence of OA (mild n=8, moderate n=4, specimens 20-31, Table 1) and sliced in
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small pieces. The cartilage was immediately treated with α-chymotrypsin, overnight as described above
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and the digested samples were stored in Eppendorf tubes and frozen at -20°C until immunoassay in
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duplicate.
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Statistical analysis
Time course study of release of C2K77 from cathepsin K digested normal equine cartilage:
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A repeated-measures linear model was used with the group (control or cathepsin K digested) as a
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between-subject factor and time points as within-subject factors. A priori contrasts were used to
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compare the pairs of means, and the alpha level for each contrast was adjusted downward using the
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sequential Benjamini-Hochberg procedure.
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Cartilage explant cytokine stimulation time course studies and comparison to unstimulated and cultured OA cartilage:
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A comparison was made between stimulated explant groups (IL-1β, IL-1β+OSM, TNF-
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α+OSM, LPS) and the control unstimulated samples at day 0. Data were transformed with the logarithm
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base 10 to normalize the distributions. A repeated-measures linear model was used with the condition
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(control, cytokine stimulation groups or OA) as a between-subject factor and time points as within-
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subject factors. A priori contrasts were used to compare pairs of means, and the alpha level for each
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contrast was adjusted downward using the sequential Benjamini-Hochberg procedure.
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C2K77 and C1,2C in fresh normal and OA cartilage: A linear model (ANOVA) with the class as a factor was used to detect differences between groups. All Statistical analyses were conducted with SAS v.9.4 (Cary, N.C.). The level of statistical
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significance was set at 5% throughout (supplementary information online).
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Results
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Development of the C2K77 ELISA assay: The range of the C2K77 ELISA was from 1 to 10,000
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ng/ml and the absorbance from 0.3 to 2.0 (Fig. 2). The limit of detection was 1 ng/ml. This corresponds
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to an antibody-peptide binding of approximately 85% (data not shown). The mean (±SD) intra-assay and
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inter-assay CV was 8.75% (± 2.78) and 6.86 % (±2.51), respectively, in the PBS BSA and 14.56%
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(±6.57) and 12.86% (±6.42), respectively, in culture media. A mean analyte spiking recovery of 99%
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(61%-126%) was observed across the assay range. The equine C2K77 antibody did not cross react with
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the corresponding human cathepsin K generated neoepitope KPGKAK (data not shown).
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Assessment of C2K77 ELISA
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Examination of healthy articular cartilage treated with and without cathepsin K to generate the type II
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collagen cleavage neoepitope:
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Digestion buffers were examined for degradation products released during the incubation with
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exogenous cathepsin K. A significant increase of C2K77 release was detected at 8h (p˂0.01) and 24 h
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(p˂0.01) in the cathepsin K treated group (Fig. 3A) compared with baseline 0h. The C2K77
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concentration was also higher in the digestion buffer of the cathepsin K digested group at 24h when
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compared to the control group (p=0.01; Fig. 3A). The remaining cartilage at 24h was further digested
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with α-chymotrypsin. The C2K77 ELISA of the α-chymotrypsin digests revealed a trend towards an
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increase in C2K77 measured cathepsin K activity in the cathepsin K treated cartilage compared to the
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control group, although not significant (Fig. 3B). All statistical values of the experiment are presented in
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supplementary data (Table S1). Prior studies demonstrated that α-chymotrypsin does not destroy the
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C2K77 neoepitope at the concentration used (data not shown).
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Cathepsin K digestion of articular cartilage: loss of proteoglycan revealed by Safranin O histology and
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in situ cleavage of type II collagen by cathepsin K detected by C2K77 immunohistochemistry:
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In healthy cartilage, there was homogenous extracellular matrix staining with Safranin O(SO;
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Fig. 4a) and an absence of immunostaining with the C2K77 antibody (Fig. 4b). In the area with a
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spontaneous partial thickness fissure there was focal loss of SO stain where C2K77 stain was observed.
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In contrast, following degradation by cathepsin K for 2h (Fig. 4c), the cartilage matrix exhibited reduced
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SO staining. The C2K77 stain increased with cathepsin K digestion (Fig. 4d) and was further augmented
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when the neoepitopes were exposed by hyaluronidase treatment, particularly deeper in the cartilage and
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in territorial sites (Figure. 4h).
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Influence of cytokines and LPS on cleavage by cathepsin K in cultured healthy equine cartilage explants
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compared to endogenous non-stimulated cathepsin K type II collagen cleavage in OA explants:
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detection of cathepsin K (C2K77) and matrix metalloproteinase (MMP) collagenase (C1,2C) activity
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with ELISA:
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C2K77 neoepitope release into culture media over time: There was an increased media
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concentration of C2K77 neoepitope at days 14 and 21 in control (p=0.003 and p=0.0001) and OA
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(p=0.003 and p=0.0005) groups compared to baseline day zero (Fig. 5A). Only IL-1β alone significantly
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increased C2K77 neoepitope release from healthy explants (p=0.0002; Fig. 5A) at day 21 compared to
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day 0. However, when compared with the untreated control group at the same time points, no significant
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differences were detected. Thus, no significant increases of C2K77 release were observed when
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cartilages were cultured with either IL-1β, IL-1β and OSM or TNF-α and OSM nor with LPS compared
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with unstimulated controls at each time point (Fig. 5C).
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There was no significant increase of C1,2C in the control group media over time compared with
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day 0. However, significant increases in the C1,2C neoepitope were observed at day 21 compared to day
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0 control cartilage with the combination of IL-1β and OSM (p=0.002), TNF-α and OSM (p˂0.0001) and
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for OA cartilages (p=0.002) (Fig. 5B). The addition of IL-1β alone or LPS did not significantly increase
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C1,2C neoepitope content in the media compared with day 0 values. An increased concentration of
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C1,2C neoepitope was also measured at days 14 (p=0.001) and 21 (p=0.002) in the IL-1β and OSM
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group compared to the unstimulated control group at the same time point (Fig.5D). C1,2C was also
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significantly increased (p=0.0004) in media of the TNF-α and OSM stimulated group compared with the
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control group, at day 21 (Fig, 5D).
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Comparison of cathepsin K (C2K77) and MMP collagenase (C1,2C) activity in freshly isolated normal
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and OA articular cartilages
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When freshly harvested cartilage was digested with α-chymotrypsin overnight at 37°C to
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solubilize the C2K77 and C1,2C neoepitopes from the cartilage matrix, there were no significant
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differences in the C2K77 and C1,2C contents between the control and OA groups. On a molar basis, the
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concentration of C2K77 is approximately 0.5 pmol/mg of cartilage and the concentration of C1,2C is
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approximately 60 pmol/mg of cartilage. Thus, C1,2C content on a cleavage neoepitope pmol basis is
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approximately 120 fold higher than C2K77 for healthy, mild and moderate OA (Fig. 6). Concentrations
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of C2K77 were similar in the control, mild and moderate OA group while C1,2C contents were
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increased in the mild and moderate OA group but the differences were not significant.
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Discussion
Normal equine cartilage digested by cathepsin K generated and released specific neoepitope-
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containing cleavage fragments that were measurable (range 0.002 to 0.005 pmol/mg of cartilage) by our
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novel C2K77 ELISA. The concentrations of cathepsin K-generated fragments reported earlier with
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another assay (C2K) for a different cathepsin K-generated collagen neoepitope in human cartilage
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samples were higher (0 to 3.5 pmol/mg of cartilage)13. The differences may reflect species differences
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related to enzymatic activity, be it enzyme expression, substrate structural differences or other reasons.
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The novel C2K77 ELISA assay had a concentration range of 1 to 10,000 ng/ml (1.4 to 14,000 nM),
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similar to that recently reported for cathepsin K digested human cartilage (0.1 to 10,000 nM)8. The
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existing C2K ELISA which we developed specifically for human type II collagen, had a range of 0.5 to
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1,000 nM5 but did not recognize any neoepitope in equine cartilage digested with cathepsin K
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(unpublished data). Furthermore, the human C2K ELISA measures a C-terminal epitope generated by
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cathepsin K cleavage of type II collagen5. The equine C2K77 ELISA assay, is based on an antibody
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directed to a different cleavage site, the most N-terminal cathepsin K cleavage site of equine type II
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collagen8, located three residues N-terminal to the cleavage position first described by Kafienah et al9 in
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human and bovine cartilage. Based on sequence data, cathepsin K may generate the same neoepitope in
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type I collagen, although there is no convincing evidence that type 1 collagen is present in any
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significant amount in healthy and early OA cartilage which would influence these analyses. In contrast,
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the C2K77 ELISA, is specific for cathepsin K cleavage of equine and not human type II collagen.
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When compared with other commercially available assays that measure type II collagen
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degradation by MMPs the range of the C2K77 assay is similar to that of C1,2C (range 30 to 10,000
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ng/ml: IBEX Technologies Inc.) but greater than either C2C (10 to 1,000 ng/ml; IBEX Technologies
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Inc.) or the C-terminal telopeptide of type II collagen (CTX-II) in serum (30 to 400 pg/ml; Nordic
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Bioscience)19. The intra- and inter-assay CVs of C2K77 were less than 10% in PBS, 1% BSA matrix
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and increased to less than 15% for culture media. CV% of 10-15% are acceptable performance targets
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for ELISA development20-22.
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The C2K77 and C1,2C assays have the capacity to determine molar concentrations of their
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respective neoepitopes although antibody affinities may vary. In an earlier study of human knee normal
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and osteoarthritic cartilages5 where another cathepsin K cleavage neoepitope of type II collagen (C2K)
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was studied we observed that the C2C neoepitope (recognized by anti- C1,2C antibodies because of
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shared sequence) was only 2-3 times higher than the concentration of the C2K neoepitope. This would
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suggest that either the C2K77 neoepitope is much more susceptible to secondary cleavage, as observed
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for the C2K epitope on extended cathepsin K digestion5, or/and that the degradation of type II collagen
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by these proteinases is different in equine cartilage.
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The lack of increase in the C2K77 neoepitope following digestion by cathepsin K, accompanied
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by the release of this neoepitope into media, suggests that C2K77 may be preferentially released from
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cartilage on digestion with cathepsin K.
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Immunohistochemical staining of the cathepsin K digested cartilage with the C2K77 antibody
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further confirmed that extracellular type II collagen of equine articular cartilage was digested on
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exposure to exogenous cathepsin K in the presence of aggrecan but that this can only be clearly detected
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by immunostaining following removal of GAG chains by hyaluronidase. An increased staining was
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observed in territorial and in pericellular sites where the digestion was more limited. As the cartilage
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employed in these studies was from adult horses, type II collagen in interterritorial sites, may be more
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resistant to cathepsin K cleavage, possibly due to increased glycation23. Our studies of degenerative cartilages failed to reveal a significant increase in the C2K77
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neoepitope compared to healthy cartilage. Yet there was a progressive increase in cathepsin K mediated
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degradation of type II collagen observed in healthy normal appearing cartilage, cultured without a
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stimulant over time. It may point to a previously unreported constant background turnover of type II
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collagen by cathepsin K in normal healthy cartilage. What is responsible for this remains unclear. None
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of the MMP stimulants we assessed in this study had the capacity to upregulate endogenous cathepsin K
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activity measured by the C2K77 assay. This was in contrast to the observed stimulation of collagenase
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induced C1,2C generation. Thus, mechanisms that may be responsible for an increase in expression and
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activity of equine articular cartilage cathepsin K in vivo remain to be determined.(please see
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Supplementary Discussion).
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We recognize that our study has limitations. We require analyses of gene expression alongside
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analyses by immunoassay of enzyme concentration in addition to measurements of enzyme activity as
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we have used here. Although the cartilage specimens were graded from mild to severe OA based on
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macroscopic appearance, it could be argued that the scoring system is not capturing early OA, when
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molecular events occur prior to cartilage matrix destruction. Additional selection of specimens based on
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histology or gene expression would have permitted the inclusion of these early OA specimens and
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perhaps have allowed the assay to detect changes at this early stage. For this initial validation of the
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C2K77 ELISA assay we elected to investigate its performance in culture media and in digests of
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articular cartilage. Additional experiments involving immunoassays using C2K77 will be required in the
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more challenging matrix of body fluids such as synovial fluid from normal and OA joints, serum and
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urine. It remains to be determined whether the low molar concentrations of this neoepitope present in
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equine articular cartilages will be detectable in vivo in synovial and other body fluids. The fact that type
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II collagen peptides are concentrated in urine23 may facilitate their measurement. A comparison with the
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neoepitope C2K assay5 in cartilage and its release from explants thereof in culture media to C2K77
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content and release would be also provide additional valuable insight so that release patterns for the N-
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terminal and C-terminal neoepitopes can be established.
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In summary cathepsin K activity involving the specific cleavage of type II collagen is measurable
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by a novel cathepsin K specific cleavage site ELISA. Its activity and regulation are clearly different
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from that of the MMP collagenases measured by the C1,2C assay.
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Supplementary discussion online.
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Acknowledgements:
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We are grateful to Alexander Emmott (Genetics Unit, Shriners Hospitals for Children, Canada.) and
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Suzanne Bourdon (IBEX, Montreal, Canada) for technical advice on ELISA development.
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Author contributions
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SL, RP and JM conceived and designed the project, interpreted data and revised the manuscript. BN
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performed the experiments and wrote the first draft of the manuscript. GB performed the statistical
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analysis. HR supervised the experiments and made figures.
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Funding
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Beatriz Noé was funded by Mathematics of Information Technology and Complex Systems (MITACS,
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Canada; https://www.mitacs.ca/en/about-mitacs). Sheila Laverty’s laboratory is currently funded by the
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National Science and Engineering Council of Canada (NSERC), and the Réseau ThéCell, Fonds de
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Recherche en Santé Québec (FRSQ).
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Conflict of interest
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None of the authors had any conflict of interest.
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References
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1. Yuan XL, Meng HY, Wang YC, Peng J, Guo QY, Wang AY, et al. Bone-cartilage interface crosstalk in osteoarthritis: potential pathways and future therapeutic strategies. Osteoarthritis Cartilage. 2014;22(8):10771089. Epub 2014/06/15. doi: 10.1016/j.joca.2014.05.023. PubMed PMID: 24928319. 2. Karsdal MA, Bay-Jensen AC, Lories RJ, Abramson S, Spector T, Pastoureau P, et al. The coupling of bone and cartilage turnover in osteoarthritis: opportunities for bone antiresorptives and anabolics as potential treatments? Ann Rheum Dis. 2014;73(2):336-348. Epub 2013/11/29. doi: 10.1136/annrheumdis-2013-204111. PubMed PMID: 24285494. 3. Bian Q, Wang YJ, Liu SF, Li YP. Osteoarthritis: genetic factors, animal models, mechanisms, and therapies. Front Biosci (Elite Ed). 2012;4:74-100. Epub 2011/12/29. PubMed PMID: 22201856. 4. Chevalier X, Richette P. Cartilage articulaire normal : anatomie, physiologie, métabolisme, vieillissement. EMC - Rhumatologie-Orthopédie. 2005;2(1):41-58. 5. Dejica VM, Mort JS, Laverty S, Percival MD, Antoniou J, Zukor DJ, et al. Cleavage of type II collagen by cathepsin K in human osteoarthritic cartilage. Am J Pathol. 2008;173(1):161-169. Epub 2008/05/31. doi: 10.2353/ajpath.2008.070494. PubMed PMID: 18511517; PubMed Central PMCID: PMCPmc2438294. 6. Poole AR, Nelson F, Dahlberg L, Tchetina E, Kobayashi M, Yasuda T, et al. Proteolysis of the collagen fibril in osteoarthritis. Biochem Soc Symp. 2003(70):115-123. Epub 2003/11/01. PubMed PMID: 14587287. 7. Poole AR, Kojima T, Yasuda T, Mwale F, Kobayashi M, Laverty S. Composition and structure of articular cartilage: a template for tissue repair. Clin Orthop Relat Res. 2001(391 Suppl):S26-33. Epub 2001/10/18. PubMed PMID: 11603710.
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8. Mort JS, Beaudry F, Theroux K, Emmott AA, Richard H, Fisher WD, et al. Early cathepsin K degradation of type II collagen in vitro and in vivo in articular cartilage. Osteoarthritis Cartilage. 2016;24(8):1461-1469. Epub 2016/04/07. doi: 10.1016/j.joca.2016.03.016. PubMed PMID: 27049030. 9. Kafienah W, Bromme D, Buttle DJ, Croucher LJ, Hollander AP. Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix. Biochem J. 1998;331 ( Pt 3):727-732. Epub 1998/04/30. PubMed PMID: 9560298; PubMed Central PMCID: PMCPmc1219411. 10. Hou WS, Li Z, Buttner FH, Bartnik E, Bromme D. Cleavage site specificity of cathepsin K toward cartilage proteoglycans and protease complex formation. Biol Chem. 2003;384(6):891-897. Epub 2003/07/31. doi: 10.1515/bc.2003.100. PubMed PMID: 12887056. 11. Bromme D, Panwar P, Turan S. Cathepsin K osteoporosis trials, pycnodysostosis and mouse deficiency models: Commonalities and differences. Expert Opin Drug Discov. 2016;11(5):457-472. Epub 2016/03/24. doi: 10.1517/17460441.2016.1160884. PubMed PMID: 27001692. 12. Vinardell T, Dejica V, Poole AR, Mort JS, Richard H, Laverty S. Evidence to suggest that cathepsin K degrades articular cartilage in naturally occurring equine osteoarthritis. Osteoarthritis Cartilage. 2009;17(3):375383. Epub 2008/09/24. doi: 10.1016/j.joca.2008.07.017. PubMed PMID: 18809344. 13. Dejica VM, Mort JS, Laverty S, Antoniou J, Zukor DJ, Tanzer M, et al. Increased type II collagen cleavage by cathepsin K and collagenase activities with aging and osteoarthritis in human articular cartilage. Arthritis Res Ther. 2012;14(3):R113. Epub 2012/05/16. doi: 10.1186/ar3839. PubMed PMID: 22584047; PubMed Central PMCID: PMCPmc3446490. 14. Billinghurst RC, Dahlberg L, Ionescu M, Reiner A, Bourne R, Rorabeck C, et al. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest. 1997;99(7):1534-1545. Epub 1997/04/01. doi: 10.1172/jci119316. PubMed PMID: 9119997; PubMed Central PMCID: PMCPmc507973. 15. Reboul P, Pelletier JP, Tardif G, Cloutier JM, Martel-Pelletier J. The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. A role in osteoarthritis. Journal of Clinical Investigation. 1996;97(9):2011-2019. PubMed PMID: PMC507274. 16. Martel-Pelletier J, Pelletier JP. Wanted--the collagenase responsible for the destruction of the collagen network in human cartilage! Br J Rheumatol. 1996;35(9):818-820. Epub 1996/09/01. PubMed PMID: 8810663. 17. Poole AR, Ionescu M, Fitzcharles MA, Billinghurst RC. The assessment of cartilage degradation in vivo: development of an immunoassay for the measurement in body fluids of type II collagen cleaved by collagenases. J Immunol Methods. 2004;294(1-2):145-153. Epub 2005/01/11. PubMed PMID: 15637808. 18. Emmott AA, Mort JS. Efficient processing of procathepsin K to the mature form. Protein Expr Purif. 2013;91(1):37-41. Epub 2013/07/13. doi: 10.1016/j.pep.2013.06.012. PubMed PMID: 23845403. 19. Duclos ME, Roualdes O, Cararo R, Rousseau JC, Roger T, Hartmann DJ. Significance of the serum CTX-II level in an osteoarthritis animal model: a 5-month longitudinal study. Osteoarthritis Cartilage. 2010;18(11):14671476. 20. Crowther JR. Validation of Diagnostic Tests for Infectious Diseases. The Elisa Guidebook. Methods in Molecular Biology. 1 ed. Totowa, NJ: Humana Press; 2000. p. 305. 21. Administration FaD. Guidance for industry : Bioanalytical Method Validation. In: Services USDoHaH, editor. Rockville, MD Food and Drug Administration; 2001. p. 5,6,10,13. 22. Thomsson O, Strom-Holst B, Sjunnesson Y, Bergqvist AS. Validation of an enzyme-linked immunosorbent assay developed for measuring cortisol concentration in human saliva and serum for its applicability to analyze cortisol in pig saliva. Acta Vet Scand. 2014;56:55. Epub 2014/09/07. doi: 10.1186/s13028-014-0055-1. PubMed PMID: 25193181; PubMed Central PMCID: PMCPMC4172964. 23. Poole AR, Ha N, Bourdon S, Sayre EC, Guermazi A, Cibere J. Ability of a Urine Assay of Type II Collagen Cleavage by Collagenases to Detect Early Onset and Progression of Articular Cartilage Degeneration: Results from a Population-based Cohort Study. J Rheumatol. 2016;43(10):1864-1870. Epub 2016/08/03. doi: 10.3899/jrheum.150917. PubMed PMID: 27481905.
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Legends
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Figure 1. Neoepitopes representing cathepsin K and collagenase cleavage sites on type II collagen
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Locations of initial cleavage events on the equine type II collagen triple helix by the action of MMP
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collagenases and cathepsin K are indicated by arrows. The peptide sequences of terminal neoepitopes
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used to prepare the antibodies employed in this study are indicated. P* represents hydroxyproline.
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Figure 2. Standard curve for the ELISA C2K77 assay
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The standard curve was generated with a synthetic C2K77 peptide. Samples were analyzed in triplicate
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and each point is the mean of the triplicates.
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Figure 3. Equine articular cartilage digested with and without cathepsin K.
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C2K77 release into media and content in cartilage matrix were measured. Fresh articular cartilage was
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digested with cathepsin K (250 nM) and digestion buffer were collected at time zero, 8 and 24 hrs. The
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enzyme was inactivated with E-64 and C2K77 epitope measured with the ELISA (A). The cartilage was
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digested with α-chymotrypsin overnight at 37°C, the enzyme was inactivated with TPCK, and an ELISA
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for C2K77 was performed (B). n =3 at 0 and 24 hours and n =2 at 8 hours. p-values are presented as
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supplementary data (Table S1).
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Figure 4. Cleavage of type II collagen in osteochondral sections in situ by Cathepsin K.
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Representative images of equine articular cartilage from metacarpal osteochondral samples, untreated (a
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& b), treated with hyaluronidase for 30 mins (e and f, g and h) or prior digested with cathepsin K (250
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nM) for 2 hours (c, d, g and h). Sections were stained with Safranin-O and Fast Green to reveal
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proteoglycan content (a, c, e and g). Site-matched sections were stained with the C2K77 antibody for
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collagen degradation by cathepsin K (b, d, f and h)
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a. A central area of focal cartilage degeneration with partial thickness fissures and adjacent normal
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appearing cartilage can be seen.
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b. Superficial increased C2K77 uptake is visible.
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c & d. Full thinkness loss of PG with a more pronounced C2K77 stain is observed.
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e & f. The hyaluronidase pretreatment reduces PG content and enhances the C2K77 signal observed in
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b.
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g & h. Full thickness hyaline cartilage staining with the C2K77 antibody. Selective C2K77 territorial
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staining can be observed in deep zone sites where digestion is limited. The hyaluronidase treatment
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enhances the C2K77 signal when compared with d.
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Scale = 500 µm
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Figure 5. Influence of cytokines and LPS on cleavage by cathepsin K in cultured healthy equine
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cartilage explants compared to endogenous non-stimulated cathepsin K type II collagen cleavage
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in OA explants: detection of cathepsin K (C2K77) and matrix metalloproteinase (MMP)
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collagenase (C1,2C) activity with ELISA
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Articular cartilages were cultured with cytokines or LPS for 21 days. Media were collected and
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replenished every 7 days. They were analyzed by ELISA for C2K77 (A, C) and for C1,2C (B, D)
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contents. *: p≤0.003. p-values are presented as supplementary data (Table S2).
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Figure 6. Comparison of cathepsin K (C2K77) and MMP collagenase (C1,2C) activity in freshly
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isolated normal and OA articular cartilages
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Articular cartilage was digested with α-chymotrypsin overnight at 37°C. After inhibition of the enzyme
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with TCPK, the C2K77 and C1,2C contents were assessed by ELISA. p-values are presented as
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supplementary data (Table S3).
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Table 1. Details of specimens employed in cathepsin K digestion, in histologie and immunohistochemistry, in cartilage explant and in
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OA
Cathepsin K Digestion
Cartilage Explant
Chymotrypsin digestion fresh cartilage
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Experiment
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Macroscopic appearance Healthy Healthy Healthy Healthy OA Healthy Healthy Healthy OA Moderate OA Moderate OA severe Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy OA Mild OA Mild OA Mild OA Mild OA Mild OA Mild OA Mild OA Mild OA Moderate OA Moderate OA Moderate OA Moderate
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Ages(years) N/A N/A 10 8 4 2 4 7 5 21 30 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
Histology and immunohistochemistry
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mistry, in cartilage explant and in α-chymotrypsin digestion of fresh and OA cartilage study.
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S1063-4584(17)31159-7
DOI:
10.1016/j.joca.2017.08.011
Reference:
YJOCA 4074
To appear in:
Osteoarthritis and Cartilage
Received Date: 20 January 2017 Revised Date:
21 August 2017
Accepted Date: 26 August 2017
Please cite this article as: Noé B, Poole AR, Mort JS, Richard H, Beauchamp G, Laverty S, C2K77 ELISA detects cleavage of type II collagen by cathepsin K in equine articular cartilage, Osteoarthritis and Cartilage (2017), doi: 10.1016/j.joca.2017.08.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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C2K77 ELISA detects cleavage of type II collagen by cathepsin K in equine articular cartilage
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Authors: Beatriz Noé^*, A. Robin Pooleǂ, John S. Mortǂ, Helene Richard^, Guy Beauchamp¶, Sheila
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Laverty^
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Affiliation : ^Comparative Orthopaedic Research Laboratory, Département de sciences cliniques,
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Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe (QC) J2S
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2M2, Canada
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ǂDivision of Orthopaedics, Department of Surgery, McGill University, Montreal, (QC), Canada
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¶ Département de Pathologie et Microbiologie Vétérinaires, Faculté de Médecine Vétérinaire,
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Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe (QC) J2S 2M2, Canada
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*Beatriz Noé’s current address is : Département de sciences cliniques, Faculté de Médecine Vétérinaire,
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Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe (QC) J2S 2M2, Canada. Tel 1-438-881-2892.
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E-mail : [email protected]
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Corresponding author: S Laverty MVB, DACVS & DECVS, Département de sciences cliniques, Faculté
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de Médecine Vétérinaire, Université de Montréal, 3200 Rue Sicotte, St-Hyacinthe (QC) J2S 2M2,
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Canada. Tel1-450 7788100. Email: [email protected]
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Abstract Objectives: Develop a species-specific ELISA for a neo-epitope generated by cathepsin K
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cleavage of equine type II collagen to: 1) measure cartilage type II collagen degradation by cathepsin K
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in vitro, 2) identify cytokines that upregulate cathepsin K expression and 3) compare cathepsin K with
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MMP collagenase activity in stimulated cartilage explants and freshly isolated normal and OA articular
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cartilages.
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Design: A new ELISA (C2K77) was developed and tested by measuring the activity of
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exogenous cathepsin K on equine articular cartilage explants. The ELISA was then employed to
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measure endogenous cathepsin K activity in cultured cartilage explants with or without stimulation by
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interleukin-1 beta (IL-1β), tumour necrosis-alpha (TNF-α), oncostatin M (OSM) and lipopolysaccharide
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(LPS). Cathepsin K activity in cartilage explants (control and osteoarthritic-OA) and freshly harvested
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cartilage (control and OA) was compared to that of MMPs employing C2K77 and C1,2C immunoassays.
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Results: The addition of Cathepsin K to normal cartilage caused a significant increase (p˂0.01) in
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the C2K77 epitope release. Whereas the content of C1,2C, that reflects MMP collagenase activity, was
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increased in media by the addition to cartilage explants of TNF-α and OSM (p˂0.0001) or IL-1β and
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OSM (p = 0.002), no change was observed in C2K77 which also unchanged in OA cartilages compared
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to normal.
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Conclusions: The ELISA C2K77 measured the activity of cathepsin K in equine cartilage which was
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unchanged in OA cartilage. Cytokines that upregulate MMP collagenase activity had no effect on
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endogenous cathepsin K activity, suggesting a different activation mechanism that requires further
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study.
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Keywords
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cathepsin K, type II collagen, ELISA, osteoarthritis, cartilage, MMPs
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Introduction Osteoarthritis (OA) is a progressive degenerative joint disease characterized by articular cartilage
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degeneration, subchondral bone remodelling, synovial inflammation and pain1. Many factors can trigger
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OA including joint trauma and overuse, aging, obesity2 and genetic predisposition3.
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Type II collagen, the most abundant collagen in the extracellular matrix of articular cartilage,
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represents 95% of the total collagen content4. The collagen fibril is composed of a triple helix of three
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identical α-chains, imparting tensile strength essential for maintaining the integrity of cartilage4. In OA,
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this collagen is progressively degraded by enzymatic digestion, essentially an irreversible event in
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cartilage degeneration in OA5.
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Few vertebrate proteases are able to cleave intact type II collagen. Following primary cleavage,
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and molecule unwinding, it becomes susceptible to additional cleavage by many extracellular proteases.
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Thus, primary cleavage is a key step in cartilage matrix degradation in OA6.
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Matrix metalloproteinase collagenases (MMPs) are involved in this primary cleavage in OA6.
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MMPs 1, 8 and 13 can cleave type II collagen near the C-terminal end: MMP 13 may be especially
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responsible in OA7. But cathepsin K, may also play an important role in cartilage degradation in OA. Cathepsin K can cleave intact type II collagen at multiple sites
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. It is a cysteine protease
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capable of degrading many cartilage matrix molecules including proteoglycans10, in the case if aggrecan
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at several sites in the G1, interglobular and chondroitin sulfate domains10. Originally found in osteoclasts,
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playing an important role in bone resorption11, it is also present in chondrocytes12. In contrast to the
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MMPs, cathepsin K cleaves type II collagen close to the N-terminus at various helical sites8. Our studies
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have revealed an increased abundance of cathepsin K, and its degradation products of cleaved type II
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collagen, adjacent to chondrocytes in OA cartilage suggesting it plays a role in matrix degradation in
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equine12 and human5, 13 joint disease. The capacity of cytokines and other molecules to stimulate MMP collagenase activity involved
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in type II collagen degradation in OA has been well studied14-16 but little is known about the regulation
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of cathepsin K. Such knowledge would help us understand its role in disease, including OA, and could
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potentially lead to the development of a biomarker of cartilage degradation in OA.
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Cathepsin K cleavage sites, have been recently identified (Fig. 1). An antibody against one such
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cleavage site (C2K77) has been raised by us in rabbits8. Immunostaining of OA cartilage sections
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revealed the antibody’s capacity to detect cathepsin K cleavage in OA cartilage8 suggesting that the
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antibody may be a suitable candidate for an ELISA immunoassay.
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We hypothesized that cathepsin K activity in cartilage is measurable by a cathepsin K specific
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cleavage neoepitope ELISA, similar to those developed for MMP collagenases14, 17. Our first objective
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was to develop a novel, competitive ELISA inhibition-assay directed against a neoepitope generated by
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the cathepsin K cleavage of equine type II collagen. Our second objective was to measure type II
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collagen degradation by cathepsin K in vitro in normal, stimulated and OA articular cartilages and
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determine whether cytokines regulate cathepsin K activity. Because it may be comparable to that of
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MMP collagenases, our third objective was to compare cathepsin K and collagenase activities using
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specific anti-neoepitope based ELISA assays in stimulated cartilage explants and also in freshly isolated
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control and OA cartilage.
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Methods
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Development of a competitive C2K77 ELISA
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C2K77 ELISA:
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The anti-neoepitope (C2K77) antibody was produced by immunizing rabbits with a synthetic
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peptide, KPGKSGGC, that includes the equine type II collagen cleavage (the most N-terminal cleavage)
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neoepitope generated by cathepsin K8. It has been shown previously8 that this antibody does not
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recognize intact alpha chains of type II collagen and does not react with uncleaved collagen. The C2K77
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immunizing peptide and the purified antibody were titrated to attain the optimal operational
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concentrations for establishment of the competitive C2K77. Sensitivity, reproducibility and recovery
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were assessed. The antibody was tested to detect specificity to equine peptide by immunoassaying 50
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ng/ml of the human C2K77 immunizing peptide. The C2K77 ELISA was then employed to detect the
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specific cathepsin K generated neoepitopes contained in type II collagen cleavage fragments, and
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released from cartilage or present in the cartilage matrix, under a variety of experimental conditions, to
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better understand the involvement of cathepsin K activity in health and pathology.
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Equine articular cartilage
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Articular cartilage was harvested from equine healthy and OA metacarpophalangeal (MCP)
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joints obtained from a local abattoir or from horses donated to the veterinary teaching hospital at the
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Université de Montréal (CHUV). For more details see Supplementary Material.
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Assessment of C2K77 ELISA by digestion of healthy articular cartilage with cathepsin K to
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generate type II collagen cleavage fragment release over time
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Healthy equine cartilages (specimens 1-4; Table 1) were chopped into small pieces and placed in
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Eppendorf tubes containing PBS. Cartilage was then incubated in 2 ml of digestion buffer at 32°C and
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agitated for 24 h with 250 nM recombinant human cathepsin K18. Aliquots (50 µl) of the digestion
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buffer were harvested at 0, 8 and 24 h. Trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-
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64) was added to each aliquot to inactivate cathepsin K. Control cartilage was similarly treated but
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without the addition of cathepsin K. Supernatants in Eppendorf tubes were stored at –20°C until
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immunoassay with the C2K77 ELISA. On termination of the cathepsin K and control digestions, the
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cartilage was collected, blotted dry, weighed and stored at -20°C. In order to solubilize the C2K77
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neoepitope in the cartilage matrix, as in previous studies5, a part of the remaining cartilage was digested
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with α-chymotrypsin. The C2K77 immunizing peptide was mixed with chymotrypsin – 1mg/ml ( BSA
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1mg/ml) for 24 hrs and then assayed to confirm that the enzyme did not digest the peptide. Alpha
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chymotrypsin was then inactivated by the addition of N-tosyl-L-phenylalanine-chloromethyl ketone
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(TPCK). The digestion buffer was analyzed in duplicate with the C2K77 ELISA.
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Demonstration that cathepsin K can cleave type II collagen in situ As part of the study it was important to determine whether we could demonstrate cleavage of
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type II collagen in situ by cathepsin K. In order to address this we digested cartilage in equine
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osteochondral sections containing normal and osteoarthritic cartilage (specimen 32; Table 1). For more
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details see Supplementary Material.
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Ability of cytokines (IL-1β, IL-1β+OSM, TNF-α+OSM) or LPS to stimulate cleavage by cathepsin
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K in cultured healthy equine cartilage explants and comparison to endogenous non-stimulated
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cathepsin K mediated type II collagen cleavage in OA explants
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Full depth articular cartilage was harvested under aseptic conditions from healthy equine MCP
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joints (n = 3) (specimens 6-8, Table 1) and from joints with OA (n=3) (specimens 9-11, Table 1). The
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cartilage was placed in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen Canada Inc.)
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containing antibiotics/anti-mycotic agents (Penicillin 385.6 units/ml, streptomycin 385.6 µg/ml and
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Amphotericin 0.963 µg/ml) for 30 mins at 37°. Two additional washing steps, 15 min per washing, were
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performed with 30 ml of DMEM containing antibiotics/anti-mycotic. The cartilage was then chopped
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into approximately 2 x 3 mm explants and again washed. The cartilage was weighed and 50-70 mg was
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randomly placed into each well of a 24-well tissue culture plates containing 1 ml of DMEM with 10%
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fetal bovine serum (FBS). The cartilage explants were pre-cultured for 2 days at 37°C in a humidified
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incubator with 5% CO2/95% air. The explants were cultured in 24 well plates and four wells were used
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per stimulation, whenever sufficient tissue was available.
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To simulate inflammatory stimuli encountered in joint disease, culture wells contained IL-1β (10
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ng/ml) (Kingfisher Biotech USA) or IL-1β and OSM (1 ng/ml and 10 ng/ml, respectively) (OSM:
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Prepotech, USA) or TNF-α and OSM (10 ng/ml and 10 ng/ml, respectively) (TNF: Preprotech, USA) or
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LPS (10 µg/ml) (Sigma-Aldrich, USA). Wells without these additives served as controls. The culture
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media were harvested and replaced every 7 days, up to day 21, stored in Eppendorf tubes and frozen at -
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20°C.
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Media were immunoassayed employing the C1,2C ELISA (IBEX Technologies Inc., Montreal,
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Quebec, Canada), an immunoassay specific for a neoepitope generated by type II collagen cleavage by
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MMP collagenases, and the C2K77 immunoassay.
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Concentrations of C2K77 and C1,2C neoepitopes in fresh normal and OA cartilage
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Cartilage was harvested from healthy equine joints (n=8; specimens 12-19, Table 1) and joints
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with macroscopic evidence of OA (mild n=8, moderate n=4, specimens 20-31, Table 1) and sliced in
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small pieces. The cartilage was immediately treated with α-chymotrypsin, overnight as described above
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and the digested samples were stored in Eppendorf tubes and frozen at -20°C until immunoassay in
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duplicate.
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Statistical analysis
Time course study of release of C2K77 from cathepsin K digested normal equine cartilage:
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A repeated-measures linear model was used with the group (control or cathepsin K digested) as a
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between-subject factor and time points as within-subject factors. A priori contrasts were used to
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compare the pairs of means, and the alpha level for each contrast was adjusted downward using the
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sequential Benjamini-Hochberg procedure.
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Cartilage explant cytokine stimulation time course studies and comparison to unstimulated and cultured OA cartilage:
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A comparison was made between stimulated explant groups (IL-1β, IL-1β+OSM, TNF-
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α+OSM, LPS) and the control unstimulated samples at day 0. Data were transformed with the logarithm
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base 10 to normalize the distributions. A repeated-measures linear model was used with the condition
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(control, cytokine stimulation groups or OA) as a between-subject factor and time points as within-
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subject factors. A priori contrasts were used to compare pairs of means, and the alpha level for each
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contrast was adjusted downward using the sequential Benjamini-Hochberg procedure.
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C2K77 and C1,2C in fresh normal and OA cartilage: A linear model (ANOVA) with the class as a factor was used to detect differences between groups. All Statistical analyses were conducted with SAS v.9.4 (Cary, N.C.). The level of statistical
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significance was set at 5% throughout (supplementary information online).
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Results
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Development of the C2K77 ELISA assay: The range of the C2K77 ELISA was from 1 to 10,000
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ng/ml and the absorbance from 0.3 to 2.0 (Fig. 2). The limit of detection was 1 ng/ml. This corresponds
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to an antibody-peptide binding of approximately 85% (data not shown). The mean (±SD) intra-assay and
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inter-assay CV was 8.75% (± 2.78) and 6.86 % (±2.51), respectively, in the PBS BSA and 14.56%
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(±6.57) and 12.86% (±6.42), respectively, in culture media. A mean analyte spiking recovery of 99%
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(61%-126%) was observed across the assay range. The equine C2K77 antibody did not cross react with
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the corresponding human cathepsin K generated neoepitope KPGKAK (data not shown).
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Assessment of C2K77 ELISA
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Examination of healthy articular cartilage treated with and without cathepsin K to generate the type II
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collagen cleavage neoepitope:
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Digestion buffers were examined for degradation products released during the incubation with
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exogenous cathepsin K. A significant increase of C2K77 release was detected at 8h (p˂0.01) and 24 h
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(p˂0.01) in the cathepsin K treated group (Fig. 3A) compared with baseline 0h. The C2K77
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concentration was also higher in the digestion buffer of the cathepsin K digested group at 24h when
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compared to the control group (p=0.01; Fig. 3A). The remaining cartilage at 24h was further digested
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with α-chymotrypsin. The C2K77 ELISA of the α-chymotrypsin digests revealed a trend towards an
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increase in C2K77 measured cathepsin K activity in the cathepsin K treated cartilage compared to the
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control group, although not significant (Fig. 3B). All statistical values of the experiment are presented in
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supplementary data (Table S1). Prior studies demonstrated that α-chymotrypsin does not destroy the
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C2K77 neoepitope at the concentration used (data not shown).
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Cathepsin K digestion of articular cartilage: loss of proteoglycan revealed by Safranin O histology and
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in situ cleavage of type II collagen by cathepsin K detected by C2K77 immunohistochemistry:
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In healthy cartilage, there was homogenous extracellular matrix staining with Safranin O(SO;
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Fig. 4a) and an absence of immunostaining with the C2K77 antibody (Fig. 4b). In the area with a
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spontaneous partial thickness fissure there was focal loss of SO stain where C2K77 stain was observed.
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In contrast, following degradation by cathepsin K for 2h (Fig. 4c), the cartilage matrix exhibited reduced
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SO staining. The C2K77 stain increased with cathepsin K digestion (Fig. 4d) and was further augmented
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when the neoepitopes were exposed by hyaluronidase treatment, particularly deeper in the cartilage and
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in territorial sites (Figure. 4h).
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Influence of cytokines and LPS on cleavage by cathepsin K in cultured healthy equine cartilage explants
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compared to endogenous non-stimulated cathepsin K type II collagen cleavage in OA explants:
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detection of cathepsin K (C2K77) and matrix metalloproteinase (MMP) collagenase (C1,2C) activity
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with ELISA:
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C2K77 neoepitope release into culture media over time: There was an increased media
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concentration of C2K77 neoepitope at days 14 and 21 in control (p=0.003 and p=0.0001) and OA
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(p=0.003 and p=0.0005) groups compared to baseline day zero (Fig. 5A). Only IL-1β alone significantly
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increased C2K77 neoepitope release from healthy explants (p=0.0002; Fig. 5A) at day 21 compared to
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day 0. However, when compared with the untreated control group at the same time points, no significant
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differences were detected. Thus, no significant increases of C2K77 release were observed when
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cartilages were cultured with either IL-1β, IL-1β and OSM or TNF-α and OSM nor with LPS compared
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with unstimulated controls at each time point (Fig. 5C).
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There was no significant increase of C1,2C in the control group media over time compared with
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day 0. However, significant increases in the C1,2C neoepitope were observed at day 21 compared to day
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0 control cartilage with the combination of IL-1β and OSM (p=0.002), TNF-α and OSM (p˂0.0001) and
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for OA cartilages (p=0.002) (Fig. 5B). The addition of IL-1β alone or LPS did not significantly increase
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C1,2C neoepitope content in the media compared with day 0 values. An increased concentration of
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C1,2C neoepitope was also measured at days 14 (p=0.001) and 21 (p=0.002) in the IL-1β and OSM
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group compared to the unstimulated control group at the same time point (Fig.5D). C1,2C was also
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significantly increased (p=0.0004) in media of the TNF-α and OSM stimulated group compared with the
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control group, at day 21 (Fig, 5D).
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Comparison of cathepsin K (C2K77) and MMP collagenase (C1,2C) activity in freshly isolated normal
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and OA articular cartilages
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When freshly harvested cartilage was digested with α-chymotrypsin overnight at 37°C to
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solubilize the C2K77 and C1,2C neoepitopes from the cartilage matrix, there were no significant
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differences in the C2K77 and C1,2C contents between the control and OA groups. On a molar basis, the
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concentration of C2K77 is approximately 0.5 pmol/mg of cartilage and the concentration of C1,2C is
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approximately 60 pmol/mg of cartilage. Thus, C1,2C content on a cleavage neoepitope pmol basis is
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approximately 120 fold higher than C2K77 for healthy, mild and moderate OA (Fig. 6). Concentrations
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of C2K77 were similar in the control, mild and moderate OA group while C1,2C contents were
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increased in the mild and moderate OA group but the differences were not significant.
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Discussion
Normal equine cartilage digested by cathepsin K generated and released specific neoepitope-
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containing cleavage fragments that were measurable (range 0.002 to 0.005 pmol/mg of cartilage) by our
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novel C2K77 ELISA. The concentrations of cathepsin K-generated fragments reported earlier with
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another assay (C2K) for a different cathepsin K-generated collagen neoepitope in human cartilage
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samples were higher (0 to 3.5 pmol/mg of cartilage)13. The differences may reflect species differences
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related to enzymatic activity, be it enzyme expression, substrate structural differences or other reasons.
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The novel C2K77 ELISA assay had a concentration range of 1 to 10,000 ng/ml (1.4 to 14,000 nM),
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similar to that recently reported for cathepsin K digested human cartilage (0.1 to 10,000 nM)8. The
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existing C2K ELISA which we developed specifically for human type II collagen, had a range of 0.5 to
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1,000 nM5 but did not recognize any neoepitope in equine cartilage digested with cathepsin K
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(unpublished data). Furthermore, the human C2K ELISA measures a C-terminal epitope generated by
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cathepsin K cleavage of type II collagen5. The equine C2K77 ELISA assay, is based on an antibody
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directed to a different cleavage site, the most N-terminal cathepsin K cleavage site of equine type II
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collagen8, located three residues N-terminal to the cleavage position first described by Kafienah et al9 in
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human and bovine cartilage. Based on sequence data, cathepsin K may generate the same neoepitope in
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type I collagen, although there is no convincing evidence that type 1 collagen is present in any
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significant amount in healthy and early OA cartilage which would influence these analyses. In contrast,
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the C2K77 ELISA, is specific for cathepsin K cleavage of equine and not human type II collagen.
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When compared with other commercially available assays that measure type II collagen
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degradation by MMPs the range of the C2K77 assay is similar to that of C1,2C (range 30 to 10,000
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ng/ml: IBEX Technologies Inc.) but greater than either C2C (10 to 1,000 ng/ml; IBEX Technologies
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Inc.) or the C-terminal telopeptide of type II collagen (CTX-II) in serum (30 to 400 pg/ml; Nordic
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Bioscience)19. The intra- and inter-assay CVs of C2K77 were less than 10% in PBS, 1% BSA matrix
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and increased to less than 15% for culture media. CV% of 10-15% are acceptable performance targets
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for ELISA development20-22.
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The C2K77 and C1,2C assays have the capacity to determine molar concentrations of their
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respective neoepitopes although antibody affinities may vary. In an earlier study of human knee normal
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and osteoarthritic cartilages5 where another cathepsin K cleavage neoepitope of type II collagen (C2K)
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was studied we observed that the C2C neoepitope (recognized by anti- C1,2C antibodies because of
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shared sequence) was only 2-3 times higher than the concentration of the C2K neoepitope. This would
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suggest that either the C2K77 neoepitope is much more susceptible to secondary cleavage, as observed
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for the C2K epitope on extended cathepsin K digestion5, or/and that the degradation of type II collagen
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by these proteinases is different in equine cartilage.
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The lack of increase in the C2K77 neoepitope following digestion by cathepsin K, accompanied
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by the release of this neoepitope into media, suggests that C2K77 may be preferentially released from
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cartilage on digestion with cathepsin K.
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Immunohistochemical staining of the cathepsin K digested cartilage with the C2K77 antibody
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further confirmed that extracellular type II collagen of equine articular cartilage was digested on
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exposure to exogenous cathepsin K in the presence of aggrecan but that this can only be clearly detected
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by immunostaining following removal of GAG chains by hyaluronidase. An increased staining was
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observed in territorial and in pericellular sites where the digestion was more limited. As the cartilage
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employed in these studies was from adult horses, type II collagen in interterritorial sites, may be more
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resistant to cathepsin K cleavage, possibly due to increased glycation23. Our studies of degenerative cartilages failed to reveal a significant increase in the C2K77
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neoepitope compared to healthy cartilage. Yet there was a progressive increase in cathepsin K mediated
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degradation of type II collagen observed in healthy normal appearing cartilage, cultured without a
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stimulant over time. It may point to a previously unreported constant background turnover of type II
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collagen by cathepsin K in normal healthy cartilage. What is responsible for this remains unclear. None
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of the MMP stimulants we assessed in this study had the capacity to upregulate endogenous cathepsin K
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activity measured by the C2K77 assay. This was in contrast to the observed stimulation of collagenase
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induced C1,2C generation. Thus, mechanisms that may be responsible for an increase in expression and
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activity of equine articular cartilage cathepsin K in vivo remain to be determined.(please see
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Supplementary Discussion).
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We recognize that our study has limitations. We require analyses of gene expression alongside
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analyses by immunoassay of enzyme concentration in addition to measurements of enzyme activity as
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we have used here. Although the cartilage specimens were graded from mild to severe OA based on
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macroscopic appearance, it could be argued that the scoring system is not capturing early OA, when
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molecular events occur prior to cartilage matrix destruction. Additional selection of specimens based on
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histology or gene expression would have permitted the inclusion of these early OA specimens and
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perhaps have allowed the assay to detect changes at this early stage. For this initial validation of the
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C2K77 ELISA assay we elected to investigate its performance in culture media and in digests of
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articular cartilage. Additional experiments involving immunoassays using C2K77 will be required in the
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more challenging matrix of body fluids such as synovial fluid from normal and OA joints, serum and
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urine. It remains to be determined whether the low molar concentrations of this neoepitope present in
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equine articular cartilages will be detectable in vivo in synovial and other body fluids. The fact that type
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II collagen peptides are concentrated in urine23 may facilitate their measurement. A comparison with the
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neoepitope C2K assay5 in cartilage and its release from explants thereof in culture media to C2K77
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content and release would be also provide additional valuable insight so that release patterns for the N-
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terminal and C-terminal neoepitopes can be established.
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In summary cathepsin K activity involving the specific cleavage of type II collagen is measurable
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by a novel cathepsin K specific cleavage site ELISA. Its activity and regulation are clearly different
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from that of the MMP collagenases measured by the C1,2C assay.
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Supplementary discussion online.
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Acknowledgements:
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We are grateful to Alexander Emmott (Genetics Unit, Shriners Hospitals for Children, Canada.) and
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Suzanne Bourdon (IBEX, Montreal, Canada) for technical advice on ELISA development.
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Author contributions
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SL, RP and JM conceived and designed the project, interpreted data and revised the manuscript. BN
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performed the experiments and wrote the first draft of the manuscript. GB performed the statistical
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analysis. HR supervised the experiments and made figures.
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Funding
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Beatriz Noé was funded by Mathematics of Information Technology and Complex Systems (MITACS,
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Canada; https://www.mitacs.ca/en/about-mitacs). Sheila Laverty’s laboratory is currently funded by the
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National Science and Engineering Council of Canada (NSERC), and the Réseau ThéCell, Fonds de
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Recherche en Santé Québec (FRSQ).
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Conflict of interest
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None of the authors had any conflict of interest.
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8. Mort JS, Beaudry F, Theroux K, Emmott AA, Richard H, Fisher WD, et al. Early cathepsin K degradation of type II collagen in vitro and in vivo in articular cartilage. Osteoarthritis Cartilage. 2016;24(8):1461-1469. Epub 2016/04/07. doi: 10.1016/j.joca.2016.03.016. PubMed PMID: 27049030. 9. Kafienah W, Bromme D, Buttle DJ, Croucher LJ, Hollander AP. Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix. Biochem J. 1998;331 ( Pt 3):727-732. Epub 1998/04/30. PubMed PMID: 9560298; PubMed Central PMCID: PMCPmc1219411. 10. Hou WS, Li Z, Buttner FH, Bartnik E, Bromme D. Cleavage site specificity of cathepsin K toward cartilage proteoglycans and protease complex formation. Biol Chem. 2003;384(6):891-897. Epub 2003/07/31. doi: 10.1515/bc.2003.100. PubMed PMID: 12887056. 11. Bromme D, Panwar P, Turan S. Cathepsin K osteoporosis trials, pycnodysostosis and mouse deficiency models: Commonalities and differences. Expert Opin Drug Discov. 2016;11(5):457-472. Epub 2016/03/24. doi: 10.1517/17460441.2016.1160884. PubMed PMID: 27001692. 12. Vinardell T, Dejica V, Poole AR, Mort JS, Richard H, Laverty S. Evidence to suggest that cathepsin K degrades articular cartilage in naturally occurring equine osteoarthritis. Osteoarthritis Cartilage. 2009;17(3):375383. Epub 2008/09/24. doi: 10.1016/j.joca.2008.07.017. PubMed PMID: 18809344. 13. Dejica VM, Mort JS, Laverty S, Antoniou J, Zukor DJ, Tanzer M, et al. Increased type II collagen cleavage by cathepsin K and collagenase activities with aging and osteoarthritis in human articular cartilage. Arthritis Res Ther. 2012;14(3):R113. Epub 2012/05/16. doi: 10.1186/ar3839. PubMed PMID: 22584047; PubMed Central PMCID: PMCPmc3446490. 14. Billinghurst RC, Dahlberg L, Ionescu M, Reiner A, Bourne R, Rorabeck C, et al. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest. 1997;99(7):1534-1545. Epub 1997/04/01. doi: 10.1172/jci119316. PubMed PMID: 9119997; PubMed Central PMCID: PMCPmc507973. 15. Reboul P, Pelletier JP, Tardif G, Cloutier JM, Martel-Pelletier J. The new collagenase, collagenase-3, is expressed and synthesized by human chondrocytes but not by synoviocytes. A role in osteoarthritis. Journal of Clinical Investigation. 1996;97(9):2011-2019. PubMed PMID: PMC507274. 16. Martel-Pelletier J, Pelletier JP. Wanted--the collagenase responsible for the destruction of the collagen network in human cartilage! Br J Rheumatol. 1996;35(9):818-820. Epub 1996/09/01. PubMed PMID: 8810663. 17. Poole AR, Ionescu M, Fitzcharles MA, Billinghurst RC. The assessment of cartilage degradation in vivo: development of an immunoassay for the measurement in body fluids of type II collagen cleaved by collagenases. J Immunol Methods. 2004;294(1-2):145-153. Epub 2005/01/11. PubMed PMID: 15637808. 18. Emmott AA, Mort JS. Efficient processing of procathepsin K to the mature form. Protein Expr Purif. 2013;91(1):37-41. Epub 2013/07/13. doi: 10.1016/j.pep.2013.06.012. PubMed PMID: 23845403. 19. Duclos ME, Roualdes O, Cararo R, Rousseau JC, Roger T, Hartmann DJ. Significance of the serum CTX-II level in an osteoarthritis animal model: a 5-month longitudinal study. Osteoarthritis Cartilage. 2010;18(11):14671476. 20. Crowther JR. Validation of Diagnostic Tests for Infectious Diseases. The Elisa Guidebook. Methods in Molecular Biology. 1 ed. Totowa, NJ: Humana Press; 2000. p. 305. 21. Administration FaD. Guidance for industry : Bioanalytical Method Validation. In: Services USDoHaH, editor. Rockville, MD Food and Drug Administration; 2001. p. 5,6,10,13. 22. Thomsson O, Strom-Holst B, Sjunnesson Y, Bergqvist AS. Validation of an enzyme-linked immunosorbent assay developed for measuring cortisol concentration in human saliva and serum for its applicability to analyze cortisol in pig saliva. Acta Vet Scand. 2014;56:55. Epub 2014/09/07. doi: 10.1186/s13028-014-0055-1. PubMed PMID: 25193181; PubMed Central PMCID: PMCPMC4172964. 23. Poole AR, Ha N, Bourdon S, Sayre EC, Guermazi A, Cibere J. Ability of a Urine Assay of Type II Collagen Cleavage by Collagenases to Detect Early Onset and Progression of Articular Cartilage Degeneration: Results from a Population-based Cohort Study. J Rheumatol. 2016;43(10):1864-1870. Epub 2016/08/03. doi: 10.3899/jrheum.150917. PubMed PMID: 27481905.
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Legends
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Figure 1. Neoepitopes representing cathepsin K and collagenase cleavage sites on type II collagen
4
Locations of initial cleavage events on the equine type II collagen triple helix by the action of MMP
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collagenases and cathepsin K are indicated by arrows. The peptide sequences of terminal neoepitopes
6
used to prepare the antibodies employed in this study are indicated. P* represents hydroxyproline.
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Figure 2. Standard curve for the ELISA C2K77 assay
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The standard curve was generated with a synthetic C2K77 peptide. Samples were analyzed in triplicate
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and each point is the mean of the triplicates.
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Figure 3. Equine articular cartilage digested with and without cathepsin K.
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C2K77 release into media and content in cartilage matrix were measured. Fresh articular cartilage was
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digested with cathepsin K (250 nM) and digestion buffer were collected at time zero, 8 and 24 hrs. The
15
enzyme was inactivated with E-64 and C2K77 epitope measured with the ELISA (A). The cartilage was
16
digested with α-chymotrypsin overnight at 37°C, the enzyme was inactivated with TPCK, and an ELISA
17
for C2K77 was performed (B). n =3 at 0 and 24 hours and n =2 at 8 hours. p-values are presented as
18
supplementary data (Table S1).
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Figure 4. Cleavage of type II collagen in osteochondral sections in situ by Cathepsin K.
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Representative images of equine articular cartilage from metacarpal osteochondral samples, untreated (a
22
& b), treated with hyaluronidase for 30 mins (e and f, g and h) or prior digested with cathepsin K (250
23
nM) for 2 hours (c, d, g and h). Sections were stained with Safranin-O and Fast Green to reveal
24
proteoglycan content (a, c, e and g). Site-matched sections were stained with the C2K77 antibody for
25
collagen degradation by cathepsin K (b, d, f and h)
26
a. A central area of focal cartilage degeneration with partial thickness fissures and adjacent normal
27
appearing cartilage can be seen.
28
b. Superficial increased C2K77 uptake is visible.
29
c & d. Full thinkness loss of PG with a more pronounced C2K77 stain is observed.
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e & f. The hyaluronidase pretreatment reduces PG content and enhances the C2K77 signal observed in
31
b.
32
g & h. Full thickness hyaline cartilage staining with the C2K77 antibody. Selective C2K77 territorial
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staining can be observed in deep zone sites where digestion is limited. The hyaluronidase treatment
34
enhances the C2K77 signal when compared with d.
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Scale = 500 µm
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Figure 5. Influence of cytokines and LPS on cleavage by cathepsin K in cultured healthy equine
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cartilage explants compared to endogenous non-stimulated cathepsin K type II collagen cleavage
39
in OA explants: detection of cathepsin K (C2K77) and matrix metalloproteinase (MMP)
40
collagenase (C1,2C) activity with ELISA
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Articular cartilages were cultured with cytokines or LPS for 21 days. Media were collected and
42
replenished every 7 days. They were analyzed by ELISA for C2K77 (A, C) and for C1,2C (B, D)
43
contents. *: p≤0.003. p-values are presented as supplementary data (Table S2).
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Figure 6. Comparison of cathepsin K (C2K77) and MMP collagenase (C1,2C) activity in freshly
46
isolated normal and OA articular cartilages
47
Articular cartilage was digested with α-chymotrypsin overnight at 37°C. After inhibition of the enzyme
48
with TCPK, the C2K77 and C1,2C contents were assessed by ELISA. p-values are presented as
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supplementary data (Table S3).
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Table 1. Details of specimens employed in cathepsin K digestion, in histologie and immunohistochemistry, in cartilage explant and in
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OA
Cathepsin K Digestion
Cartilage Explant
Chymotrypsin digestion fresh cartilage
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Experiment
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Macroscopic appearance Healthy Healthy Healthy Healthy OA Healthy Healthy Healthy OA Moderate OA Moderate OA severe Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy OA Mild OA Mild OA Mild OA Mild OA Mild OA Mild OA Mild OA Mild OA Moderate OA Moderate OA Moderate OA Moderate
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Ages(years) N/A N/A 10 8 4 2 4 7 5 21 30 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
Histology and immunohistochemistry
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Specimens 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
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mistry, in cartilage explant and in α-chymotrypsin digestion of fresh and OA cartilage study.
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