Osteoclast-associated receptor blockade prevents articular cartilage destruction via chondrocyte apoptosis regulation

Abstract

Osteoarthritis (OA), primarily characterized by articular cartilage destruction, is the most common form of age-related degenerative whole-joint disease. No disease-modifying treatments for OA are currently available. Although OA is primarily characterized by cartilage destruction, our understanding of the processes controlling OA progression is poor. Here, we report the association of OA with increased levels of osteoclast-associated receptor (OSCAR), an immunoglobulin-like collagen-recognition receptor. In mice, OSCAR deletion abrogates OA manifestations, such as articular cartilage destruction, subchondral bone sclerosis, and hyaline cartilage loss. These effects are a result of decreased chondrocyte apoptosis, which is caused by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in induced OA. Treatments with human OSCAR-Fc fusion protein attenuates OA pathogenesis caused by experimental OA. Thus, this work highlights the function of OSCAR as a catabolic regulator of OA pathogenesis, indicating that OSCAR blockade is a potential therapy for OA.

Fig. 1. OSCAR expression in mice and humans is upregulated in articular chondrocytes in OA.

a Immunostaining for OSCAR protein (top) and mRNA levels (bottom-right) in articular chondrocytes of Oscar^−/− and WT mice. Error bars represent mean ± S.E.M. of n = 10 mice. Scale bar = 50 μm (top), 20 μm (bottom). b IHC (top) and qRT-PCR (bottom-right) analyses of OSCAR in articular cartilage of DMM surgery mice (compared to sham-operated mice). Error bars represent mean ± S.E.M. of n = 10 mice. Scale bar = 50 μm (top), 20 μm (bottom). c Representative images show immunostaining (top) and quantitative analyses (bottom-right) of OSCAR in intact and damaged regions of human OA articular cartilage. Error bars represent mean ± S.E.M. of n = 10 patient samples. Scale bar = 200 μm (top), 50 μm (bottom). Student’s two-tailed t test was used for statistical analysis, with p values displayed in figure.

Fig. 2. Oscar^-−/− mice exhibit reduced OA pathogenesis.

a–d Experimental OA was examined by safranin-O staining (a) and scoring of OA parameters, including articular cartilage destruction (OARSI grade) (b), SBP thickness (c), and ratio of hyaline cartilage (HC) to the calcified cartilage (CC) (d) in WT and Oscar^−/− mice. Oscar^−/− and WT mice underwent sham or DMM surgery for 8 weeks. Scale bar = 50 μm. e Representative 3D reconstructed micro-CT images and quantitative analysis of mouse tibia subchondral bone plates at 8 weeks post DMM surgery compared with that of the sham control. Scale bars = 1000 μm. Error bars represent mean ± S.E.M. of n = 10 mice (b–e). f, g qRT-PCR analysis of articular cartilage tissue of WT and Oscar^−/− mice subjected to sham or DMM surgery with error bars representing mean ± S.E.M. of n = 10 mice. h, i IHC analyses of MMP3, MMP13 and ADAMTS5 (h), aggrecan and COL2A1 (i) in OA articular cartilage from DMM surgery mice compared with sham-operated mice (n = 10). Scale bar = 50 μm. Two-way ANOVA was performed followed by Sidak’s Multiple Comparison’s test, with p values indicated in figure.

Fig. 3. OSCAR regulates OA pathogenesis via TRAIL-induced articular chondrocyte apoptosis.

a Selection of putative Oscar targets involved in OA pathogenesis. Venn diagram indicating the number of genes attenuated in articular cartilage Oscar^−/− mice after DMM surgery (left). Hypergeometric p value measuring the significance of enrichment is depicted. Gene-wise scaled gene expression patterns of 1270 common genes in each sample are shown (right). b Functional annotations significantly enriched for 1270 common genes. Hypergeometric tests were performed using the hallmark gene annotation in MsigDB, yielding enrichment scores, defined as −log₁₀ (FDR). c Simplified apoptotic signaling pathway altered by Oscar deficiency. Each gene is colored with the DIF score, defined as the extent to which gene expression is altered by Oscar deficiency. A negative DIF indicates transcriptional suppression in Oscar^−/− mice. The network was visualized by Cytoscape v3.7 software. d, e qRT-PCR (d) and IHC (e) analyses of TRAIL in OA articular cartilage from DMM surgery mice compared to sham-operated mice. Scale bar = 50 μm. f, g mRNA levels (f) and immunostaining (g) for OPG in articular cartilage tissue from sham surgery or DMM surgery mice. Scale bar = 50 μm. h, i Apoptotic articular chondrocytes were detected and quantified by TUNEL assay. Scale bar = 25 μm. Error bars represent mean ± S.E.M. of n = 10 mice (d–g, i). Two-way ANOVA was performed followed by Sidak’s Multiple Comparison’s test, with p values indicated in figure.

Fig. 4. OSCAR–collagen binding co-stimulates IL-1β-induced apoptotic signaling in articular chondrocytes.

a Caspase activity in mouse articular chondrocytes treated with IL-1β (5 ng mL⁻¹) and collagenase (Coll, 50 U mL⁻¹). b, c qRT-PCR analysis (b) and western blotting (c) in mouse articular chondrocytes. Chondrocytes were either untreated or transfected with control siRNA (C-siRNA) (100 nM) or siRNAs specific for mouse Oscar and exposed to IL-1β (5 ng mL⁻¹) and collagenase (50 U mL⁻¹) for 48 h. d Articular chondrocyte viability was quantified by MTT assay. Error bars represent mean ± S.E.M. of n = 5 wells. e Caspase-8 and caspase-3 activity was measured using the respective assay kits. f, g qRT-PCR analysis (f) and western blotting (g) in mouse articular chondrocytes. Chondrocytes were treated with hIgG or hOSCAR-Fc (10 μg ml⁻¹) with collagenase and exposed to IL-1β for 48 h. h Apoptotic articular chondrocytes were detected and quantified by TUNEL assay. Scale bar = 100 μm. i, j qRT-PCR analysis (i) and western blotting (j) in mouse articular chondrocytes treated with 10 μΜ OSCAR-binding triple-helical peptide before IL-1β treatment. Articular chondrocytes were treated with hOSCAR-Fc for 48 h. Error bars shown in a, b, e, f, h and i are mean ± S.E.M. for n = 4 independent experiments. One-way ANOVA was performed followed by Dunnett’s Multiple Comparison’s test (a, i) and Tukey’s Multiple Comparison’s test (b, d, e, f, h), with p values indicated in figure.

Fig. 5. OSCAR-Fc blocks OA pathogenesis in mice.

WT mice subjected to sham or DMM surgery were IA-injected with human IgG as a control or hOSCAR-Fc (2 mg kg⁻¹) to block OSCAR in joint tissue. Articular cartilage sections were subjected to safranin-O staining (a) and quantitative analysis of OARSI grade (b), SBP thickness (c), and ratio of hyaline cartilage (HC) to calcified cartilage (CC) (d). Scale bar = 50 μm. Error bars represent mean ± S.E.M. of n = 10 mice (b–d). e–g IHC analyses of MMP3, MMP13, and ADAMTS5 (e), aggrecan and COL2A1 (f), and TRAIL and OPG (g) in articular cartilage tissue from sham surgery or DMM surgery mice (n = 10). Scale bar = 50 μm. h, i Apoptotic articular chondrocytes were detected and quantified by TUNEL assay. Scale bar = 25 μm. Error bars represent mean ± S.E.M. of n = 10 mice (i). Two-way ANOVA was performed followed by Sidak’s Multiple Comparison’s test, with p values indicated in figure.