Zoledronate promotes ECM degradation and apoptosis via Wnt/β-catenin

Abstract

This study examined the potential mechanism of zoledronate on interleukin (IL)-1β-induced temporomandibular joint osteoarthritis (TMJOA) chondrocytes, using IL-1β-induced rabbit immortalized mandibular condylar chondrocytes cultured with zoledronate. Cell viability, apoptosis, mRNA, and protein expression of relevant genes involved in extracellular matrix (ECM) degradation, apoptosis, and Wnt/β-catenin signaling were examined. The involvement of the Wnt/β-catenin signaling was examined using Wnt/β-catenin inhibitor (2-(4-(trifluoromethyl)phenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-ol (XAV-939)) and activator lithium chloride (LiCl). Aggrecan and type II collagen were downregulated by zoledronate, especially with 100 nM for 48 h (p < 0.01), consistently with the upregulation of A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) (p < 0.001), matrix metalloprotease-9 (MMP-9) (p < 0.01), caspase-3 (p < 0.001) and downregulation of proliferating cell nuclear antigen (PCNA) (p < 0.01). The apoptotic rate increased from 34.1% to 45.7% with 100 nM zoledronate for 48 h (p < 0.01). The effects of zoledronate on ADAMTs4 (p < 0.001), MMP-9 (p < 0.001), caspase-3 (p < 0.001), and PCNA (p < 0.01) were reversed by XAV-939, while LiCl increased caspase-3 expression (p < 0.01). In conclusion, zoledronate enhances IL-1β-induced ECM degradation and cell apoptosis in TMJOA chondrocytes. Wnt/β-catenin signaling might be involved in this process, but additional studies are necessary to determine the exact involvement of Wnt/β-catenin signaling in chondrocytes after zoledronate treatment.

Keywords: Wnt signaling pathway; apoptosis; extracellular matrix; osteoarthritis; temporomandibular joint; zoledronate.

Figure 1

Expression levels of ECM-related genes in IL-1β-treated rabbit IMCCs. Expression levels of (a) aggrecan, (b) type II collagen, (c) ADAMTS-4, (d) ADAMTS-5, and (e) MMP-9 were analyzed by qRT-PCR. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ns: not significant.

Figure 2

The morphological and abundance changes of TMJOA chondrocytes treated with different concentrations and times of ZOL (scale bar = 200 µm).

Figure 3

Expression levels of ECM-related genes and proteins after treatment with 10 or 100 nM ZOL in TMJOA chondrocytes. (a) Aggrecan, (b) type II collagen, (c) ADAMTS-4, (d) ADAMTS-5, and (e) MMP-9 were analyzed by qRT-PCR. (f) ECM-related proteins were detected by western blotting. Black bars represent the control group, and gray bars represent the 10 ng/mL IL-1β group. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups linked by the line; all other comparisons are not significant.

Figure 4

Expression levels of apoptosis- and proliferation-related genes, proteins, and percentage of apoptotic cells after treatment with 10 or 100 nM ZOL in IMCCs. (a) Caspase-3, (b) Caspase-9, and (c) PCNA were analyzed by qRT-PCR. (d) Caspase-3 and PCNA proteins were detected by western blotting. (e) Flow cytometry analysis for the apoptotic cells. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups linked by the line; all other comparisons are not significant.

Figure 5

Determination of the concentrations of XAV-939 and LiCl. Cell viability was measured by the MTT assay after XAV-939 (a) or LiCl (b) and 100 nM ZOL treatment of TMJOA cells for 48 h.

Figure 6

Involvement of Wnt/β-catenin signaling in the effects of ZOL to TMJOA degradation. Relative mRNA expression levels of (a) aggrecan, (b) ADAMTS-4, (c) MMP-9, (d) Caspase-3, and (e) PCNA were analyzed by qRT-PCR. (f) Western blotting analysis of those proteins. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 between the two groups linked by the line; all other comparisons are not significant.