Tenascin-C fragments are endogenous inducers of cartilage matrix degradation

Nidhi Sofat, Saralili Dipa Robertson, Monika Hermansson, Jonathan Jones, Philip Mitchell, Robin Wait, Nidhi Sofat, Saralili Dipa Robertson, Monika Hermansson, Jonathan Jones, Philip Mitchell, Robin Wait

Abstract

Cartilage destruction is a hallmark of osteoarthritis (OA) and is characterized by increased protease activity resulting in the degradation of critical extracellular matrix (ECM) proteins essential for maintaining cartilage integrity. Tenascin-C (TN-C) is an ECM glycoprotein, and its expression is upregulated in OA cartilage. We aimed to investigate the presence of TN-C fragments in arthritic cartilage and establish whether they promote cartilage degradation. Expression of TN-C and its fragments was evaluated in cartilage from subjects undergoing joint replacement surgery for OA and RA compared with normal subjects by western blotting. The localization of TN-C in arthritic cartilage was also established by immunohistochemistry. Recombinant TN-C fragments were then tested to evaluate which regions of TN-C are responsible for cartilage-degrading activity in an ex vivo cartilage explant assay measuring glycosaminoglycan (GAG) release, aggrecanase and matrix metalloproteinase (MMP) activity. We found that specific TN-C fragments are highly upregulated in arthritic cartilage. Recombinant TN-C fragments containing the same regions as those identified from OA cartilage mediate cartilage degradation by the induction of aggrecanase activity. TN-C fragments mapping to the EGF-L and FN type III domains 3-8 of TN-C had the highest levels of aggrecan-degrading ability that was not observed either with full-length TN-C or with other domains of TN-C. TN-C fragments represent a novel mechanism for cartilage degradation in arthritis and may present new therapeutic targets for the inhibition of cartilage degradation.

Figures

Fig. 1
Fig. 1
Western blot analysis of cartilage extracts from patients using an anti-TN-C antibody. A total of 1 μg protein was run per well and membranes developed with alkaline phosphatase (AP) substrate for 1 min each. Sample identification: 1 29 (F), lower leg, amputation; 2 17 (F), lower limb, osteosarcoma; 3 45 (M), lower limb, amputation; 4 72 (M), hip OA; 5 68 (M), hip OA; 6 72 (M), knee OA; 7 75 (F), knee OA; 8 71 (M), hip OA; 9 70 (F), knee OA; 10 66 (F), knee OA; 11 45 (M), hip, RA; 12 69 (F), hip, RA. b Equivalent samples used as in (a) with 1 μg protein per well stained with Coomassie Brilliant Blue
Fig. 2
Fig. 2
Domain composition of TN-C fragments identified from arthritic cartilage. Human TN-C has a modular structure with repeating units: the TA attachment (heptad) domain at the N-terminus, 14.5 EGF-L repeats, 8–17 FN type III repeats (conserved type III repeats numbered 1–8 are present in all isoforms with splice variants labelled by their designated letter format) and a fibrinogen (FBG) globe at the C-terminus. The table shows the composition of TN-C fragments isolated from arthritic cartilage in our study
Fig. 3
Fig. 3
Distribution of TN-C in human OA cartilage. a–d are images from 2 OA subjects stained with anti-TN-C antibody. e–h are the same OA subjects stained with an isotype control IgG mouse antibody. Zones of cartilage are delineated using the following abbreviations—S superficial, M midzone, D deep zone. Bars indicate section size of 10 μM or 50 μM
Fig. 4
Fig. 4
TN-C fragments induce cartilage degradation in normal articular cartilage. a Recombinant TN-C fragments used for cartilage stimulation were run on SDS–PAGE. All proteins were loaded at 1 μg each and stained with silver. b TN-C fragments at 1 μM were used to stimulate porcine cartilage explants for 2 days. The culture medium was harvested and tested for the presence of GAG using the DMMB dye assay (n = 3). c, d Equal volumes of conditioned media (50 μl) from the experiment shown in b were deglycosylated and western blots performed for aggrecanase-cleaved neoepitopes using the BC-3 antibody (c) and the BC-14 antibody (d). Loading controls were medium alone, c, used as a negative control and IL-1α, which served as a positive control
Fig. 5
Fig. 5
a Porcine cartilage was stimulated with recombinant TN-C fragments with a concentration range of 0.01–1 μM. After 2 days, conditioned medium was harvested and GAG release measured using the DMMB assay (n = 3). b Normal human cartilage (obtained from a 69-year-old subject with a femoral neck fracture) was stimulated with recombinant TN-C fragments at 1 μM each. Conditioned medium was harvested after 2 days and GAG release measured using the DMMB assay (n = 3)
Fig. 6
Fig. 6
Scheme for role of TN-C and TN-C fragments in OA pathology

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