Protective role of frizzled-related protein B on matrix metalloproteinase induction in mouse chondrocytes

All experiments were made on explants or primary chondrocytes extracted from 3-day-old to 6-day-old newborn litters from FrzB ^−/− or wild-type mice [18]. All procedures were in accordance with the European Directive N886/609 and were performed according to the protocols approved by French and European ethics committees for animal use and care (Comité Régional d’Ethique en Expérimentation Animale N°3 de la région Ile de France).

The procedure for compressive loading of mouse costal cartilage explants was as described previously [30]. Briefly, explants were harvested from rib cages of 4-day-old to 6-day-old newborn mice. Samples were cleaned, divided into segments, pooled and weighed for further normalization; each sample consisted of around 30 to 40 mg costal cartilage. The explants were allowed to rest for about 20 hours in 3 ml serum-free medium (Dulbecco’s modified Eagle’s medium supplemented with 0.1% bovine serum albumin and 30 mM Hepes). They were washed before they underwent 6-hour dynamic compression in 1.5 ml of the same fresh medium (sinusoidal wave-form 0 to 1 MPa at 0.5 Hz) by the Biopress system (Flexercell International, Dunn Labortechnick GmbH, Asbach, Germany). Control explants were kept in unloaded conditions. After the application of the mechanical regimen, supernatants and cartilage explants were collected and stored immediately at −80°C. Our results are expressed as fold-induction versus the uncompressed explant controls.

Primary chondrocytes were isolated from articular cartilage of 4-day-old to 6-day-old newborn mice as described previously [31]. After 1 week of expansion, the cells were placed in serum-free conditions for 24 hours (0.1% bovine serum albumin; Sigma Aldrich, Saint-Quentin Fallavier, France) and then treated for 24 hours in serum-free medium supplemented with 0, 10, 100 or 1,000 pg/ml interleukin (IL)-1β (PeproTech, Tebu-Bio, Le Perray-en-Yvelines, France). Our results are expressed as fold-induction versus the nontreated controls.

RNA was extracted from chondrocytes cultured with and without IL-1β by use of the RNeasy minikit (Qiagen, Courtaboeuf, France); for the RNA extraction from compressed and uncompressed cartilage explants, Proteinase K (Qiagen) was first added to remove proteins as suggested for tissue samples by the manufacturer. Reverse transcription was performed on 1 μg RNA for monolayer cultured cells and on 100 to 200 ng RNA for explants using the Omniscript kit (Qiagen). Relative quantification of genes involved use of the LC480 LightCycler Real Time PCR system (Roche Applied Science, Meylan, France) and the Go Taq QPCR master mix (Promega, Charbonnières les Bains, France). mRNA levels of MMP-3 and MMP-13, β-catenin (Ctnnb1) and Lef1 were normalized to that of hypoxanthine–guanine phosphoribosyltransferase (Hprt), used as the internal standard.

The primes used (sense and antisense) were as follows: Mmp-3, s-TGAAAATGAAGGGTCTTCCGG and as-GCAGAAGCTCCATACCAGCA; Mmp-13, s-GATGGCACTGCTGACATCAT and as-TGTAGCCTTTGGAACTGCTT; Ctnnb1, s-GCAGCAGCAGTTTGTGGA and as-TGTGGAGAGCTCCAGTACACC; Lef1, s-TCCTGAAATCCCCACCTTCT and as-TGGGATAAACAGGCTGACCT; and Hprt, s-AGGACCTCTCGAAGTGT and as-ATTCAAATCCCTGAAGTACTCAT.

The amount of MMP-3 and MMP-13 released by chondrocytes in response to IL-1β was assessed in the culture supernatants. For total mouse MMP-3 secretion we used a commercially available enzyme-linked immunosorbent assay kit (R&D Systems, Lille, France). For total mouse MMP-13 secretion we performed western blot analysis as described previously [32] with rabbit polyclonal antibody for MMP-13 (H-230; Santa Cruz Biotechnology, Tebu-Bio). Densitometry analysis of immunoblots involved use of Multi Gauge software (Fujifilm, Paris, France).

The amount of glycosaminoglycan and global MMP activity was measured in the culture supernatants of compressed cartilage explants. Released glycosaminoglycan was assessed by reaction with dimethylmethylene blue [33]. Shark chondroitin sulfate was used as a standard. MMP activity was assessed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ synthetic fluorogenic substrate (Bachem, Weil am Rhein, Germany) in continuous assays [34]. Results were normalized to milligram wet weight cartilage and concentration of proteins in the culture supernatant.

Intracellular proteins were extracted from chondrocytes cultured with and without IL-1β. Assay of total mouse β-catenin involved western blot analysis with rabbit polyclonal antibody for β-catenin (Cell Signaling, Danvers, MA, USA). A reprobing with anti-β-actin antibodies (AC-15; Sigma Aldrich) served as a loading control. Densitometry analysis of immunoblots involved use of Multi Gauge software (Fujifilm). The ratio of β-catenin band intensity to the β-actin band intensity was calculated.

Data are expressed as the mean ± standard error of the mean and were analyzed by the use of Mann–Whitney nonparametric tests. Three to four independent experiments were performed. P values and number of experiments (n) are indicated in the figure legends.

Cartilage explants from FrzB ^−/− and wild-type mice were stimulated by dynamic compression for 6 hours. As previously published by our group, load induced cartilage degradation in the explants [35]. Cartilage degradation was revealed by a net increase in glycosaminoglycan release (2.8-fold, P = 0.014; Figure 1A) and MMP activity (4.5-fold, P = 0.014; Figure 1B). At baseline, cartilage degradation was not different between the FrzB^−/− and wild-type samples (data not shown). Load-induced glycosaminoglycan release was similar in FrzB^−/− and wild-type samples but load-induced increase in MMP activity was slightly enhanced in FrzB^−/− cartilage explants compared with wild-type explants (1.6-fold, P = 0.17; Figure 1B). The lack of FrzB thus tends to enhance the catabolic response of chondrocytes in situ, in response to mechanical stress.

Primary cultures of articular chondrocytes from FrzB^−/− and wild-type mice were stimulated with increasing doses of IL-1β for 24 hours (10, 100 and 1000 pg/ml). IL-1β dose-dependently induced Mmp-13 gene expression (up to 15-fold, P = 0.05; Figure 2A). At the higher dose, IL-1β-induced Mmp-13 gene expression was enhanced in FrzB^−/− chondrocytes compared with wild-type chondrocytes (P = 0.05). Of note, in basal conditions Mmp-13 gene expression was similar in FrzB^−/− and wild-type chondrocytes (0.012 ± 0.008 vs. 0.020 ± 0.008 arbitrary units, respectively). At protein level, MMP-13 release was measured by western blot analysis (Figure 2B). MMP-13 release was not modulated by the lower doses of IL-1β, but was significantly increased in response to 1,000 pg/ml (P = 0.014). This increase in MMP-13 release tended to be slightly enhanced in FrzB^−/− chondrocytes compared with wild-type chondrocytes (1.5-fold, P = 0.17). Similarly, IL-1β dose-dependently induced Mmp-3 gene expression (up to 500-fold, P = 0.05; Figure 3A). At the higher dose, IL-1β-induced Mmp-3 gene expression tended to be enhanced in FrzB^−/− chondrocytes compared with wild-type chondrocytes (P = 0.10). As for Mmp-13, Mmp-3 gene expression was similar in FrzB^−/− and wild-type chondrocytes in basal conditions (0.0021 ± 0.0010 vs. 0.0017 ± 0.0004 arbitrary units, respectively). At protein level, MMP-3 release was assessed by enzyme-linked immunosorbent assay (Figure 3B). MMP-3 was not detected in untreated conditions, but only in response to the higher doses of IL-1β (P = 0.05). MMP-3 release in response to 100 pg/ml IL-1β was enhanced in FrzB^−/− chondrocytes compared with wild-type (2.1-fold, P = 0.10). The absence of FrzB thus tends to enhance the catabolic response of cultured chondrocytes, in response to a proinflammatory stress. These results are parallel to the enhanced catabolic response observed in loaded FrzB^−/− cartilage explants.

We wondered whether the enhancement in the response of FrzB^−/− chondrocytes to IL-1β and load was associated with a deregulation in Wnt/β-catenin signaling. In basal conditions, Ctnnb1 gene expression (coding β-catenin) was comparable between FrzB^−/− cultured chondrocytes and wild-type chondrocytes (0.62 ± 0.22 vs. 0.95 ± 0.12 arbitrary units, respectively). Of note, similar results were observed for the Wnt/β-catenin target gene Lef1 (0.031 ± 0.009 for wild-type chondrocytes vs. 0.018 ± 0.013 for FrzB^−/− chondrocytes). These data are in accordance with the previous transcriptome analysis of the bone–cartilage unit of FrzB^−/− mice: at baseline in vivo, the Ctnnb1 mRNA level was not different between the FrzB^−/− and wild-type strains [36].

Ctnnb1 gene expression was not affected by IL-1β in wild-type cultured chondrocytes (Figure 4A). In IL-1β-treated FrzB^−/− chondrocytes, Ctnnb1 gene expression was induced 4.3-fold compared with untreated chondrocytes (P = 0.014). Ctnnb1 gene expression was thus markedly higher in IL-1β-treated FrzB^−/− chondrocytes compared with wild-type chondrocytes (3.6-fold, P = 0.057). In parallel, Lef1 gene expression was induced 6.2-fold in IL-1β-treated FrzB^−/− chondrocytes compared with untreated chondrocytes (P = 0.05) and the Lef1 mRNA level was clearly higher in IL-1β-treated FrzB^−/− chondrocytes compared with wild-type chondrocytes (13-fold, P = 0.05; Figure 4A). Analysis of β-catenin accumulation at the protein level did not strictly correlate this transcriptional regulation (Figure 4B). IL-1β tended to decrease the β-catenin content of wild-type cultured chondrocytes (20% less, P = 0.057), whereas no modulation was observed in FrzB^−/− chondrocytes. The β-catenin content was thus higher in IL-1β-treated FrzB^−/− chondrocytes compared with wild-type chondrocytes (1.4-fold, P = 0.10). IL-1β only slightly modulates Wnt/β-catenin signaling in our model, but FrzB-dependent deregulation was observed in IL-1β-treated chondrocytes.

Concerning the catabolic response of FrzB^−/− cartilage to load, analysis of Ctnnb1 gene expression showed very similar results (Figure 4C). The Ctnnb1 mRNA level was not affected by compression in wild-type explants. In compressed FrzB^−/− samples, Ctnnb1 gene expression was induced 2.7-fold compared with uncompressed samples (P = 0.05). The modulation in Wnt/β-catenin signaling in response to load was thus slightly enhanced in FrzB^−/− cartilage (P = 0.20; Figure 4C).

Overall, these results suggest that the enhancement in the response of FrzB^−/− chondrocytes to IL-1β and load may be associated with an overstimulation of the canonical Wnt pathway.

We have demonstrated that load-induced MMP activity was enhanced in FrzB^−/− cartilage explants. In addition, load stimulated Ctnnb1 gene expression in FrzB^−/− explants. These results suggest that the load-induced catabolic response of chondrocytes may be in part mediated by the canonical Wnt pathway. The involvement of Wnt signaling in osteoblast responsiveness to mechanical stimulation was proven in various studies [37‐42]. In particular, TOPGAL reporter mice showed that the canonical Wnt/β-catenin pathway was activated by mechanical stress in vivo and in cultured osteoblasts [43]. Furthermore, increasing basal β-catenin levels was shown to enhance the effects of mechanical stress [44]. Chondrocyte responses towards mechanical stimulation have been less studied. However, pressure-induced mechanical stress triggered β-catenin tyrosine phosphorylation in cultured chondrocytes [45], thereby probably releasing the molecule from adherens junctions and increasing its availability for intracellular signaling. Partial β-catenin nuclear translocation was also observed in response to tensile strain [27]. Furthermore, there was an additive effect of load and Wnt-3a on β-catenin translocation and on upregulation of Mmp-3 gene expression [27]. Of interest, a downregulation of FrzB was observed in human and mouse cartilage explants in response to mechanical injury, suggesting a de-repression of the canonical Wnt pathway [46]. Wnt/β-catenin signaling may thus be part of the signaling response leading to excessive catabolism and cartilage degradation in response to abnormal loading and FrzB may have a protective role on load-induced increase in MMP activity.

We demonstrated that IL-1β-mediated increases in Mmp-3 and Mmp-13 gene expression and protein release were enhanced in FrzB^−/− chondrocytes. Similarly, IL-1β stimulated Ctnnb1 and Lef1 gene expression and β-catenin accumulation in FrzB^−/− chondrocytes. Canonical Wnt pathway activation may thus enhance chondrocyte responsiveness to IL-1β. These results suggest a crosstalk between the canonical Wnt pathway, which includes β-catenin and FrzB, and the IL-1 pathway, which stimulates MMP expression in chondrocytes. In accordance with our results, activation of β-catenin signaling in cultured chondrocytes by Wnt-3a treatment potentiated IL-1β-mediated loss of proteoglycans [25]. Conversely, inhibition of β-catenin signaling by the use of Lef1 siRNA downregulated IL-1β-mediated increase in Mmp-13 gene expression [29]. Sost, a biologically active inhibitor of β-catenin signaling in chondrocytes, downregulated the IL-1α-mediated increase in Mmp-13 gene expression in cartilage explants and also reduced the loss of proteoglycans [47]. An alternative suggestion is that IL-1β treatment may induce production of canonical Wnt such as Wnt-7b, which in turn would activate β-catenin signaling [48]. In surprising contrast, in human chondrocytes β-catenin signaling was found to counteract IL-1β-mediated increase in MMP-3 and MMP-13 expressions [48]. In conclusion, the canonical Wnt pathway may be part of mechanisms leading to excessive catabolism in response to inflammatory stress and FrzB may have a protective role on IL-1-mediated increase in MMP expression in mouse chondrocytes.

In OA, cartilage breakdown is due to cleavage of matrix molecules in response to abnormal mechanical stress and to some degree of inflammation. Because our results suggest that FrzB may have a protective role on load-mediated and IL-1-mediated catabolic processes in mouse chondrocytes, we speculate that FrzB may have a protective role in OA. Our results are consistent with the increased cartilage loss observed in models of arthritis in FrzB^−/− knockout mice [18]. Moreover, gene expression of Mmp-3 was upregulated in the cartilage of FrzB^−/− mice with mBSA-induced arthritis compared with wild-type mice. However, FrzB^−/− knockout mice did not develop spontaneous OA, and they did not show aberrant Mmp gene expressions in basal conditions, except for a twofold increase for MMP-3 [36]. FrzB may thus be involved in OA progression rather than OA onset. Although FrzB may interact directly with MMP-3 [18], its protective role is probably linked with a deregulation of the Wnt pathways. The recent identification of FrzB as a blocker of hypertrophic differentiation in articular cartilage [22] promotes the hypothesis of a protective role of FrzB in OA through the Wnt-mediated regulation of hypertrophic maturation.