Kindlin-2 loss in condylar chondrocytes causes spontaneous osteoarthritic lesions in the temporomandibular joint in mice

Abstract

The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint (TMJ) osteoarthritis (OA); however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4 (Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2 and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore, Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.

Fig. 1

The high protein expression level of Kindlin-2 in mandibular condylar chondrocytes in mice. a Representative images of safranin O & fast green (SO&FG, left panel) and immunofluorescent (IF, right panels) staining of mouse TMJ sections. Higher-magnification images (red dashed boxes) are shown in lower panels. Scale bar: 50 μm. b Quantification of Kindlin-1-, 2-, and -3-positive cells in condylar cartilage. N = 3 mice per group. ***P < 0.001. c A schematic diagram illustrating the experimental design. d IF staining for Kindlin-2 expression in control or cKO TMJs at 3 months post TM injections. Scale bar: 50 μm. e Percentages of Kindlin-2-expressing cells in mandibular condylar cartilage and articular disc, respectively. Results are expressed as mean ± standard deviation (s.d.). n = 8 mice per group. ***P < 0.001; ns not significant, TM tamoxifen

Fig. 2

Kindlin-2 deficiency causes condylar cartilage lesions in TMJ in adult mice. a SO&FG staining of control or cKO TMJs at 12 weeks after TM treatments. Blue dashed boxes indicate the higher-magnification images in right panels. Scale bar: 50 μm. b SO&FG staining of control or cKO TMJs at 16 weeks after TM treatments. Blue dashed boxes indicate the articular cartilage area and white dashed boxes indicate the subchondral bone area. Green arrows indicate the hypertrophic articular chondrocytes. Red arrows indicate the loss of Safranin O-positive cartilage. Orange arrows indicate the ectopic cartilage formation. Blue arrows indicate the new woven bone formation within the hypertrophic chondrocyte areas. Black arrowheads indicate the fissures on condylar cartilage surface. Yellow arrows indicate the appearance of disorganized rounded chondrocytes. Scale bar: 50 μm. c Quantitative analyses of the Osteoarthritis Research Society International (OARSI) score. Results are expressed as mean ± standard deviation (s.d.). N = 8 mice per group. **P < 0.01; ***P < 0.001. d Quantitative analyses of Safranin O-positive areas in TMJ sections from control and cKO mice. Results are expressed as mean ± standard deviation (s.d.). n = 8 mice per group. TM tamoxifen

Fig. 3

Kindlin-2 deficiency causes subchondral bone damage and ectopic bone formation in mouse TMJ. a Representative micro-computed tomography (μCT) sections of TMJ from control and cKO mice at 16 weeks after TM treatments (left panels) and three-dimensional (3D) reconstructions of the condyles (right panels). Scale bar, 1.5 mm. Red arrowheads indicate ectopic bone formation in cKO TMJ. Green arrowheads indicate the subchondral bone damage in cKO TMJ. b–f Quantitative μCT analysis of the bone mineral density (BMD), bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N) of the mandibular condyles. n = 8 mice per group. ***P < 0.001; ns not significant

Fig. 4

Kindlin-2 loss causes condylar chondrocyte hypertrophy and ECM degradation in condylar cartilage. a IF staining for expression of Aggrecan, Col2a1, Prg4, Col10a1, Runx2, and Mmp13 in control or cKO TMJs at 12 weeks post TM treatments. Red dashed boxes indicate the higher-magnification images in right panels. Scale bar: 50 μm. b–g Quantitative data of Aggrecan- (b), Col2a1- (c), Prg4- (d), Col10a1- (e), Runx2 (f), and Mmp13-expressing cells (g) in mandibular condylar cartilages of the two groups. Results are expressed as mean ± standard deviation (s.d.). n = 8 mice per group. ***P < 0.001

Fig. 5

Kindlin-2 loss decreases chondrocyte proliferation and induces chondrocyte apoptosis in condylar cartilage. a Fluorescent staining of Ki67, TUNEL, cleaved Caspase 3, p-Ampk, p-Akt and p-Erk in control or cKO TMJ sections at 12 weeks after TM treatments. Right panels show the higher-magnification images (red dashed boxes). Scale bar: 50 μm. b–g Quantitative data of a. n = 8 mice per group. h Western blotting. Protein extracts isolated from cytosol, mitochondria, or whole cells of cultured ATDC5 cells after transfection of negative control siRNA (si-NC) or Kindlin-2-targeting siRNA (si-K2). i Western blotting analyses of protein extracts from ATDC5 cells after transfection of either Kindlin-2-expressing vector (K2) or empty vector (EV). j Cell proliferation rate normalized to the EV group. All in vitro experiments were independently repeated at least three times with similar results. Results are expressed as mean ± standard deviation (s.d.). **P < 0.01; ***P < 0.001

Fig. 6

Kindlin-2 expression is downregulated in TMJs in aged mice. a Representative SO&FG and IF staining of TMJ sections from healthy adult (4 months) and aged (18–22 months) mice. Higher-magnification images (red and green dashed boxes) are shown in lower panels. Scale bar: 50 μm. b OARSI score. c Safranin O-positive areas. d Percentage of Kindlin-2-positive cells in TMJ sections from adult and aged mice. Results are expressed as mean ± standard deviation (s.d.). n = 10 mice per group. ***P < 0.001