Prevention of Injury-Induced Osteoarthritis in Rodent Temporomandibular Joint by Targeting Chondrocyte CaSR

Abstract

Traumatic joint injuries produce osteoarthritic cartilage manifesting accelerated chondrocyte terminal differentiation and matrix degradation via unknown cellular and molecular mechanisms. Here we report the ability of biomechanical stress to increase expression of the calcium-sensing receptor (CaSR), a pivotal driver of chondrocyte terminal differentiation, in cultured chondrogenic cells subjected to fluid flow shear stress (FFSS) and in chondrocytes of rodent temporomandibular joint (TMJ) cartilage subjected to unilateral anterior cross-bite (UAC). In cultured ATDC5 cells or TMJ chondrocytes, FFSS induced Ca²⁺ loading and CaSR localization in endoplasmic reticulum (ER), casually accelerating cell differentiation that could be abrogated by emptying ER Ca²⁺ stores or CaSR knockdown. Likewise, acute chondrocyte-specific Casr knockout (KO) prevented the UAC-induced acceleration of chondrocyte terminal differentiation and matrix degradation in TMJ cartilage in mice. More importantly, local injections of CaSR antagonist, NPS2143, replicated the effects of Casr KO in preventing the development of osteoarthritic phenotypes in TMJ cartilage of the UAC-treated rats. Our study revealed a novel pathological action of CaSR in development of osteoarthritic cartilage due to aberrant mechanical stimuli and supports a therapeutic potential of calcilytics in preventing osteoarthritis in temporomandibular joints by targeting the CaSR. © 2018 American Society for Bone and Mineral Research.

Keywords: CALCIUM-SENSING RECEPTOR; CHONDROCYTE DIFFERENTIATION; DENTAL MALOCCLUSION; OSTEOARTHRITIS; TEMPOROMANDIBULAR JOINT.

Fig. 1.

Effects of FFSS (16dyn/cm² for 1hr) on expression of cell differentiation marker and expression and localization of CaSR protein in ERs in cultured rat TMJ chondrocytes with or without Ca²⁺, TG (10⁻⁶M), or NPS2143 (10⁻⁵M). (A) mRNA expression profiles by quantitative real-time PCR (qPCR) showed the ability of FFSS to upregulate the expression of terminal differentiation markers (Mmp13, ALP, Osteocalcin, and Runx2) and CaSR and downregulate the expression of early differentiation makers (Aggrecan, Col-II and PPR). These effects were abrogated by removal of extracellular Ca²⁺ or treatments with TG or NPS2143. The mRNA expression levels were normalized to the expression of housekeeping GAPDH and presented as fold-increase over control without FFSS treatment. (B,C) Immunoblotting of (B) total and (C) ER proteins with anti-CaSR antibody demonstrated the ability of FFSS to increase the expression of CaSR protein and its localization in ERs and the ability of Ca²⁺-free medium, TG, and NPS2143 to abrogate these changes. β-actin and G6Pase were used as loading controls for total and ER protein, respectively, and for normalization to calculate the expression ratios in the histograms. n=4 separate cultures. Control, cultured cells without FFSS or other treatment. Values are presented as the mean±SD. **p<0.01, *p<0.05 between groups as specified by top horizontal bars in each panel.

Fig. 2.

Effects of FFSS (16dyn/cm² for 1hr) on (A) CaSR localization, (B) Ca²⁺ loading, and (C) organelle swelling in ERs of rat TMJ chondrocytes cultured with or without extracellular Ca²⁺, TG (10⁻⁶M), or NPS2143 (10⁻⁵M). (A) Dual-fluorescence staining with anti-CaSR (in green) and anti-glucose-6-phosphatase (anti-G6Pase, in red) antibodies showed the ability of FFSS to increase CaSR localization in ERs and the ability of Ca²⁺-free medium, TG or NPS2143 to abrogate this effect. (B) Dual-fluorescence staining with Fluo-8 (in green) and ER-tracker (in red) showed the ability of FFSS to increase [Ca²⁺] in ERs. This effect was abrogated by treatments of the cells with Ca²⁺-free medium or TG, but not with NPS2143. n=4 separate cultures. Bar=5μ,m. (C) Representative transmission electron microscopy (TEM) images showed the ability of FFSS to induce swelling of mitochondria and ERs. These morphological effects by FFSS could be abrogated by treatments Ca²⁺-free medium or TG, but not with NPS2143. Control, cultured cells without FFSS or other treatment.

Fig. 3.

The impact of Casr gene knockout on FFSS-induced changes in (A) Ca²⁺ loading, (B, C) CaSR localization in ERs, (D) expression of cell differentiation markers, and (E) organelle swelling in primary chondrocytes cultured from knee joints of Casr^flox/flox mice. Induction of gene KO was performed by transduction of Cre protein in chondrocyte cultures for 24 hrs before the application of FFSS. (A) Dual-fluorescence staining of Ca²⁺ and ERs by Fluo-8 (in green) and ER-tracker (in red) respectively, showed inability of Casr KO to prevent the FFSS-induced Ca²⁺ loading in ERs. (B) Dual-fluorescence staining and (C) immunoblotting of CaSR (in green) and G6Pase (in red) showed the ability of Casr KO to abrogate the FFSS-induced CaSR localization in ERs. Bar=5μm. (D) mRNA expression profiles by qPCR showed the ability of Casr KO to block the impact of FFSS on the expression of early and terminal differentiation markers. Values are presented as the mean±SD. **p<0.01, *p<0.05 between groups as specified by top horizontal bars in each panel. (E) TEM images showed the inability of Casr KO to affect the FFSS-induced swelling of mitochondria and ERs. n=4 separate cultures.

Fig. 4.

UAC induced CaSR expression, but suppressed PPR expression in condylar cartilage of rat and/or mouse TMJs. UAC was applied in rats (A-E) and in mice (F). (A) Immunohistochemical staining showed increased % of CaSR-positive chondrocytes and increased localization of the receptor in intracellular compartments, presumably ERs of the cells in TMJ cartilage of UAC vs control rat s. n=6 rats per group. (B) qPCR analyses showed increased CaSR mRNA level and reduced PPR mRNA expression in UAC vs control cartilage. Immunoblotting showed (C) increased expression of CaSR protein in the ER extracts and (D) reduced expression of total PPR protein lysates from UAC vs Control cartilage. (E) TEM images showed profound swelling of mitochondria and ERs in chondrocytes of UAC vs Control cartilage in rats. n=6 rats per group. (F) Immunohistochemical staining also showed increased % of CaSR-positive chondrocytes and increased localization of CaSR in intracellular compartments of the cells in cartilage of UAC vs control mice. n=6 mice per group. Bars in panels (A) and (F), 10μm or 20μm in digitally enlarged pictures. Values are presented as the mean±SD. **p<0.01 and *p<0.05 between UAC and Control groups.

Fig. 5.

Effects of Tamoxifen (Tam)-induced Casr gene KO on chondrocyte functions in TMJ cartilage of mice without (Control) or with UAC. The Casr gene KO was induced by 5 daily intraperitoneal (IP) Tam injections in ^Tam-CartCasr^flox/flox mice at 8 weeks of age, followed by 3 weeks of UAC. Three control groups -- Casr^flox/flox, Casr^flox/flox injected with Tamoxifen (Casr^flox/flox+Tam), or ^Tam-CartCasr^flox/flox without Tamoxifen injections --, which did not have their Casr genes ablated, were subjected to the same UAC regimen. (A) Safranin O staining (in red) showed retention of proteoglycan content and thickness of TMJ-cartilage and decreased OA grade in ^Tam-CartCasr^flox/flox mice injected with Tamoxifen vs the other 3 control groups with or without UAC treatment. Bar=100μm. n=6 mice per group. (B) qPCR analyses of mRNA and (C) immunoblotting of Col-II and Aggrecan protein extracted from the TMJ-cartilage of the above mice showed inability of UAC to reduce the expression of early differentiation markers and to increase terminal differentiation makers in ^Tam-CartCasr^flox/flox mice injected with Tamoxifen vs other 3 controls with or without UAC. Eighteen TMJ-cartilages were pooled into 3 samples (6 cartilage per sample) for qPCR and immunoblotting analysis. All values in (B) and (D) were normalized to the Casr^flox/flox Control. Values are presented as the mean±SD. **p<0.01, *p<0.05 between groups as specified by top horizontal bars in each panel.

Fig. 6.

Effects of intra-synovial injections of CaSR antagonist (NPS2143) and agonist (Cinacalcet) on morphology (A), gene (B) and protein expression (C) in rat TMJ-cartilage without (Control) or with UAC stimulation. Test compounds (50 μl of 100 nM stock) and vehicle injections were performed on 10 weeks old rats every other day 2 days for 4 weeks. UAC began 4 weeks before drug injections and continued for another 4 weeks during drug treatments. (A) Safranin O staining (in red) showed the ability of NPS2143 to increase proteoglycan content and thickness of TMJ-cartilage and decreased OA grade, the ability of Cinacalcet to suppress these parameters in both control and UAC groups when compared to corresponding vehicle-injected group. Bar=100μm. n=6 rats per group. (B) qPCR analyses of mRNA and (C) immunoblotting analysis of Col-II and Aggrecan protein extracted from the TMJ-cartilage of the above mice showed the ability of NPS2143 to increase the expression of early differentiation markers and to suppress expression of terminal differentiation makers in both control and UAC groups when compared to corresponding vehicle-injected group and vice versa for effects of Cinacalcet. Six TMJ-cartilages were pooled into 3 samples (2 cartilage per sample) for qPCR and immunoblotting analysis. All values in (B) and (D) were normalized to the Control-Vehicle. Values are presented as the mean±SD. **p<0.01, *p<0.05 between groups as specified by top horizontal bars in each panel.

Fig. 7.

Schema for the effects of CaSR on biomechanical stress-induced chondrocyte terminal differentiation.