Wnt and RUNX2 mediate cartilage breakdown by osteoarthritis synovial fibroblast‐derived ADAMTS‐7 and ‐12

Abstract Failure of therapeutic approaches for the treatment of osteoarthritis (OA) based on the inhibition of metalloproteinases, might be because of their constitutive expression in homeostasis, together with their network complexity. The knowledge of this network would contribute to selective target pathological conditions. In this sense, blockade of mediators produced by neighbouring joint cells, such as synovial fibroblasts (SF), would prevent cartilage damage. Thus, we studied the contribution of ADAMTS‐7 and ‐12 from SF to cartilage oligomeric matrix protein (COMP) degradation, and the signalling pathways involved in their expression. We report for the first time in SF, the involvement of ERK‐Runx2 axis and Wnt/β‐catenin signalling in ADAMTS‐12 and ADAMTS‐7 expressions, respectively, with the subsequent consequences in COMP degradation from cartilage extracellular matrix. After stimulation with IL‐1β or fibronectin fragments, we showed that ERK inhibition decreased Runx2 activation and ADAMTS‐12 expression in OA‐SF, also reducing Fn‐fs‐induced COMP degradation. Blockage of Wnt signalling by DKK1 reduced ADAMTS‐7 and COMP degradation in OA‐SF as well. In addition, Wnt7B expression was induced by IL‐1β and by itself, also increasing ADAMTS‐7. Our results could contribute to the development of disease‐modifying OA drugs targeting ADAMTS‐7 and ‐12 for the prevention of extracellular matrix components degradation like COMP.

proteases in the osteoarthritic degradation of the ECM, including matrix metalloproteinases (MMPs) and several ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), such as the aggrecanases ADAMTS-4 and -5. 2,3 Given the limited regeneration capacity of the articular cartilage, prevention of its damage, results crucial to reduce disease severity. In this sense, the control of inflammation and proliferation of the synovial membrane in OA patients could contribute to cartilage protection.
In rheumatoid arthritis (RA) SF are main players involved in joint destruction, but little is known about their contribution to the pathology of OA. It is known that SF release mediators that contribute to joint destruction and to the chronicity of inflammation. Although metalloproteinases have been identified as targets of therapeutic intervention, they probably failed because of their role in homeostasis, remodelling and wound healing or might be because of the diversity of the metalloproteinases network and the complexity of their interrelations. 4 The knowledge of the location and function of this intricate network could contribute to the design of selective targets to prevent cartilage destruction.
We have recently reported the involvement of SF in the production of ADAMTS-4, -5, -7 and -12, and their contribution to ECM damage. 5 The participation of aggrecanases in cartilage destruction is well known, but the specific contribution of ADAMTS-7 and -12 is less established. ADAMTS-7 and -12 digest cartilage oligomeric matrix protein (COMP), 6-8 a non-collagenous component of cartilage.
COMP is a protein of the ECM that contributes to the maintenance of cartilage stability through its interaction with other components such as aggrecan, type II collagen or fibronectin. Moreover, the signalling pathways regulating these ADAMTS are poorly understood. Here we studied whether Runx2 and Wnt/β-catenin, two signalling pathways involved in chondrocytes metabolism and OA pathology, [9][10][11] are implicated in the production of ADAMTS-7 and -12 in SF. Moreover, we analysed the role of interleukin-1β (IL-1β), and fibronectin fragments (Fn-fs), two inflammatory mediators present in OA joints, as inducers of ADAMTS expression. 5 Finally, we studied the involvement of ADAMTS-7 and -12, and their signalling pathways in the regulation of COMP degradation in the cartilage-SF interaction.

| RNA extraction and RT-qPCR
Synovial fibroblasts were seeded in 100-mm petri dishes (3 × 10 5 cells/dish) and cultured in serum-free DMEM in the presence or absence of 10 ng/mL IL-1β (ImmunoTools, Friesoythe, Germany), Wnt3A, Wnt7B and β-actin (Applied Biosystems). Assays were performed in triplicate. Results were normalized to β-actin and plotted relative to the basal, using the ∆∆ cycle threshold method for quantification. In some cases, results were normalized to β-actin and directly showed as mRNA expression (×10 4 ).

| COMP assay in cartilage-SF cultures
Release of COMP from OA cartilage was measured in the supernatants of SF cultures over cartilage explants. 13 OA joint cartilages from four patients were provided by the Rheumatology Service at Complejo Hospitalario Universitario A Coruña (CHUAC, A Coruña, Spain). Fixed diameter (6 mm) sections were collected from adjacent areas to the injured cartilage. Samples were frozen at −80°C and stored until testing. One explant per well was attached to a 96-well plate. HD-or OA-SF were added drop-wise on top of the cartilage surface (2 × 10 4 SF per explant). After 3 hours of incubation, wells were filled with DMEM in the presence or absence of 10 µM PD98059 or 200 ng/mL DKK1 for 1 hour, followed by treatment with 10 ng/mL IL-1β or 10 nM 45 kDa Fn-fs. Cultures were continued for 7 days, and treatments were renewed every two days.
Culture supernatants were collected for detection of COMP using a Quantikine® Human COMP Immunoassay (R&D Systems).

| Statistical analysis
Statistical analysis was performed using GraphPad Prism software version 6 (GraphPad Inc, San Diego, CA). Data were subjected to normality test (Kolmogórov-Smirnov test) and equal variance test (F-test). Differences were assessed using Student's two-tailed t test for two group comparisons, or one-way analysis of variance (anova) for more than two groups, with a Bonferroni post hoc test for comparisons between each two groups. Results are presented as mean ± SEM. P-values <0.05 were considered statistically significant.

| Protein expression of ADAMTS-7 and -12 in HD-and OA-SF
To validate at protein level our previous results in ADAMTS-7 and -12 mRNA transcripts, 5 we performed ELISA analysis. Our data in culture supernatants confirmed that ADAMTS-7 release was higher in OA than in HD-SF ( Figure 1A), while ADAMTS-12 levels were similar in OA and HD-SF ( Figure 1B).

| Runx2 and Wnt signalling in ADAMTS-12 and -7 expressions
Runx2 and Wnt/β-catenin pathways are involved in the ECM remodelling. 14-16 As we previously reported the implication of the proinflammatory mediators IL-1β and Fn-fs in ADAMTS-7 and -12 production, 5 we now evaluated the involvement of Runx2 and Wnt/β-catenin signalling in the expression of ADAMTS-7 and -12 by SF stimulated with these two proinflammatory mediators. To this end, we treated the cells with PD98059 or SB203580 as inhibitors of ERK and p38 MAPK, respectively, two signalling pathways involved in Runx2 activation, 17,18 and with DKK1, a well-known inhibitor of the canonical Wnt signalling. 19,20 No differences were observed when cells were obtained from HD ( Figure S2). Together these data point to the involvement of ERK-Runx2 axis in the expression of ADAMTS-12 in OA-SF.

| Wnt signalling is implicated in the ADAMTS-7 expression
DKK1 treatment decreased ADAMTS-7 mRNA as well as protein levels in OA-SF ( Figure 4B), while only mRNA expression was reduced in HD ( Figure S3B). We did not observe any effect in cells treated with MAPK inhibitors ( Figure 4A, Figure S3A). Thus, these results suggest the involvement of Wnt/β-catenin pathway in the expression of ADAMTS-7 induced by IL-1β or Fn-fs in SF. We next studied whether these two pro-inflammatory mediators modulated Wnt ligands expression. Firstly, we investigated whether SF expressed Wnt3A and Wnt7B, two members of the canonical Wnt signalling associated with OA physiopathology. 21,22 SF did not express Wnt3A (data not shown), while Wnt7B mRNA transcripts were detected in cells from HD and OA patients with a higher expression in OA-SF ( Figure 5A). Moreover, IL-1β, but not Fn-fs, induced Wnt7B expression ( Figure 5B). In addition, Wnt7B also increased its own expression ( Figure 5B), as well as ADAMTS-7 mRNA transcripts in OA-as well as in HD-SF ( Figure 5C). with Fn-fs was the only able to decrease COMP in the supernatants from cartilage explants cultured with HD-SF ( Figure S4).

| D ISCUSS I ON
In an inflammatory context, although OA is primarily characterized by chondrocytes degradation, other cells including SF also intervene through the activation of different biochemical pathways that trigger the breakdown of the cartilage ECM mediated by proteinases. 5,23 F I G U R E 2 Activation of Runx2 in OA-SF. A, Time-course of Runx2 activation after 5, 15 and 30 minutes, and 1 and 2 hours of 10 ng/mL IL-1β or 10 nM 45 kDa Fn-fs treatment. B, Runx2 activation was measured in nuclear extracts by TransAM after 1 hour of treatment with inhibitors PD98059 (iERK) or SB203580 (ip38), followed by stimulation with IL-1β for 60 minutes, or 45 kDa Fn-fs for 30 minutes (n = 4 per experimental group). Data are presented as the percentage of activation relative to the basal (mean ± SEM of duplicate determinations). Dashed horizontal line represents the basal value. *P < 0.05, ***P < 0.001 vs IL-1β, # P < 0.05, ## P < 0.01 vs Fn-fs ADAMTS activity is essential in normal ECM remodelling processes.
However, in OA dysregulation of metalloproteinases contributes to disease progression. 2,4 The role of collagenases such as MMP-13, and aggrecanases (ADAMTS-4 and -5) has been well documented. 2,5,24 Emergent studies highlight the role of ADAMTS-7 and -12 in inflammatory and rheumatic diseases, including RA and OA, atherosclerosis and cancer. [25][26][27][28] However, their function and signalling pathways involved in their expression, triggering the damage of non-collagenous components of the ECM during OA, have not been well established yet. Here we report that protein release of ADAMTS-7 in SF from OA patients is higher than in HD, whereas no differences were observed in ADAMTS-12. These data agree with our previous findings on their mRNA levels. 5 Osteoarthritis is a multifactorial pathology where many inflammatory and degradative mediators play essential roles. In this sense, IL-1β is considered one of the main pleiotropic and proinflammatory cytokines in OA. 16,29 Besides, cartilage ECM degradation products are also relevant in the pathology. 30 Amongst them, increased levels F I G U R E 3 ADAMTS-12 expression in OA-SF after inhibition of Runx2 and Wnt/β-catenin signalling. SF were treated with inhibitors PD98059 (iERK), SB203580 (ip38) or DKK1 for 1 hour, followed by treatment with IL-1β or Wnt is a main regulator of joint remodelling, involved in cartilage and bone embryonic development. 15,16 Canonical Wnt pathway, which includes β-catenin signalling, has also been widely implicated in OA pathogenesis. 11,16,22,43,44 DKK1 is an upstream inhibitor of Wnt pathway, 46 whose expression negatively correlates with OA progression. 36 Here we report that DKK1 treatment reduces IL-1β or Fn-fs-mediated ADAMTS-7 expression, indicating an essential role of Wnt/β-catenin signalling pathway in ADAMTS-7 production in SF.
Then we analysed the expression of two Frizzled receptor ligands of the canonical Wnt signalling implicated in the OA pathology.
Wnt3A is a well-known ligand, whose involvement in OA has been previously described in chondrocytes, 15,22,47 Regarding Wnt7B, its up-regulation has been described in OA and RA, with consequences in the pathology. 43 The differences observed between OA-and HD-SF should be taken into account to specifically target these ADAMTS under

ACK N OWLED G EM ENTS
We are grateful to all patients and the collaborating clinicians for their participation in this study. R.P.G. is the guarantor of this work and, such as, had full access to all the data in the study and