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.

Keywords: ADAMTS-12; ADAMTS-7; COMP; Runx2; Wnt signalling; extracellular matrix; osteoarthritis; synovial fibroblasts.

Figure 1

Constitutive protein expression of ADAMTS‐7 and ‐12 in HD‐ and OA‐SF. Protein secretion of ADAMTS‐7 (A) and ‐12 (B) was determined by ELISA in culture supernatants (n = 4 per experimental group). Data are presented as mean ± SEM of duplicate determinations. All comparisons with a p‐value less than 0.05 are shown. *P < 0.05 OA vs HD

Figure 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

Figure 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 45 kDa Fn‐fs for 24 hours (n = 4 per experimental group). (A, B) left. mRNA expression of ADAMTS‐12 was measured by RT‐qPCR. Data are presented as mean ± SEM of four independent samples analysed in triplicate (see Section 2) relative to the basal. (A, B) right. Secretion of ADAMTS‐12 was determined by ELISA in culture supernatants. Data are presented as mean ± SEM of duplicate determinations. Dashed horizontal lines represent the basal values. *P < 0.05, ***P < 0.001 vs IL‐1β; ^# P < 0.05, ^### P < 0.001 vs Fn‐fs

Figure 4

ADAMTS‐7 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 45 kDa Fn‐fs for 24 hours (n = 4 per experimental group). (A, B) left. mRNA expression of ADAMTS‐7 was measured by RT‐qPCR. Data are presented as mean ± SEM of triplicate determinations (see Section 2) relative to the basal. (A, B) right. Secretion of ADAMTS‐7 was determined by ELISA in culture supernatants. Data are presented as mean ± SEM of duplicate determinations. Dashed horizontal lines represent the basal values. *P < 0.05, **P < 0.01 vs IL‐1β; ^## P < 0.01, ^### P < 0.001 vs Fn‐fs

Figure 5

Expression of Wnt7B in HD‐ and OA‐SF. (A, B) mRNA expression of Wnt7B was measured by RT‐qPCR in HD‐ and OA‐SF (HD, n = 5 per experimental group; OA, n = 7 per experimental group). A, Constitutive mRNA expression of Wnt7B. B, mRNA expression of Wnt7B after treatment with IL‐1β, 45 kDa Fn‐fs or Wnt7B for 24 hours. C, mRNA expression of ADAMTS‐7 was measured by RT‐qPCR in HD‐ and OA‐SF after treatment with Wnt7B for 24 hours (n = 4 per experimental group). ^& P < 0.05 OA vs HD; *P < 0.05, ***P < 0.001 vs Basal. Values are presented as mean ± SEM of triplicate determinations (see Section 2)

Figure 6

Blockage of COMP release in cultures of OA‐SF over cartilage explants. OA‐SF were seeded over cartilage explants and treated with IL‐1β or Fn‐fs for 7 days, with or without previous inhibition with DKK1 or PD98059 (iERK). COMP was detected by a Quantikine^® Human COMP Immunoassay in culture supernatants (n = 4 per experimental group). A, Time‐course of COMP at 3, 5 and 7 days at basal conditions. Data are presented as the percentage of 3 days (mean ± SEM of quadruplicate determinations). B, COMP concentration at day 7 of IL‐1β‐stimulation with DKK1 or iERK. Data are presented as the percentage relative to IL‐1β‐stimulation (mean ± SEM of quadruplicate determinations). C, COMP concentration at day 7 of 45 kDa Fn‐fs‐stimulation with DKK1 or iERK. Data are presented as the percentage relative to Fn‐fs‐stimulation (mean ± SEM of quadruplicate determinations). ^& P < 0.05, ^&&& P < 0.001 vs 7 days; **P < 0.01 vs IL‐1β, ^# P < 0.05 vs Fn‐fs

Figure 7

Schematic model of IL‐1β‐ and Fn‐fs‐mediated ADAMTS‐12 and ‐7 stimulation by Runx2 and Wnt/β‐catenin signalling modulation in OA‐SF. Synovial inflammation and cartilage ECM degradation are key events in OA pathology. SF and other cells in the joint produce proinflammatory mediators including IL‐1β. Fn‐fs are released to the microenvironment as consequence of ECM degradation. Binding of IL‐1β and Fn‐fs to their receptors, IL‐1 receptor (IL‐1R), and integrins (α, β) and Toll‐like receptors (TLRs), respectively, activates MEK‐ERK‐Runx2 axis. This activation converges in the transcription of ADAMTS‐12, with the subsequent degradation of COMP. Inhibition of MEK, and consequently ERK, with PD98059 (iERK), decreases Runx2 activation, ADAMTS‐12 transcription and COMP degradation from cartilage ECM. IL‐1β and Fn‐fs also activates Wnt/β‐catenin signalling, promoting β‐catenin binding to its nuclear cofactors TCF/LEF (T‐cell factor/lymphoid enhancer factor), and inducing ADAMTS‐7 expression, also converging in COMP cleavage from cartilage ECM. Activation of Wnt/β‐catenin by IL‐1β is mediated by induction of Wnt7B, which increases its own expression as well. Mechanisms involved in the stimulation of this signalling pathway by Fn‐fs are unknown. DKK1 inhibits this signalling pathway by blocking Wnt‐binding to its receptor, thus decreasing ADAMTS‐7 transcription and COMP degradation