HomeScienceKindlin-2 loss in condylar chondrocytes causes spontaneous osteoarthritic lesions in the temporomandibular...

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

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Kindlin-2 is strongly detected in mandibular condylar chondrocytes in mice

We determined the expression pattern of three different Kindlin proteins in mouse TMJ. TMJ specimens were collected from 3-month-old C57BL/6 male mice and histological and immunofluorescent (IF) staining analyses were performed. Our data revealed a high protein expression level of Kindlin-2 in mandibular condylar chondrocytes in TMJ (Fig. 1a, b). Conversely, Either Kindlin-1 or Kindlin-3 was almost undetectable in condylar chondrocytes (Fig. 1a). Notably, Kindlin-1 was also detected in subchondral bone tissues.

Fig. 1
figure 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

Genetic ablation of Kindlin-2 in condylar chondrocytes at adult stage induces multiple spontaneous OA-like phenotypes in TMJ

We next investigated whether Kindlin-2 expression is essential in condylar chondrocytes. We crossed Kindlin-2fl/fl mice with the AggrecanCreERT2 transgenic mice to produce Kindlin-2fl/fl; AggrecanCreERT2 (referred to as K2fl/fl; AggrecanCreERT2) mice (Fig. 1c). Sixteen 8-week-old male K2fl/fl; AggrecanCreERT2 mice were intraperitoneally injected with tamoxifen (TM) (100 mg·kg−1 body weight per day, 5 injections) for conditional deletion of Kindlin-2 gene in aggrecan-expressing chondrocytes (hereinafter referred to as cKO) (Fig. 1c). Of note, another 16 age-matched male K2fl/fl; AggrecanCreERT2 mice were administrated with corn oil and served as control group. At 3 and 4 months post tamoxifen treatments, the mice were killed and TMJ specimens were collected (n = 8 mice/group for each time point). IF staining analyses confirmed that the protein expression of Kindlin-2 was markedly downregulated in condylar chondrocytes, but not in cells of the articular disc, in cKO TMJs relative to control TMJs (Fig. 1d, e).

We next performed safranin O & fast green staining on TMJ sections and the results showed that cKO mice displayed early signatures of OA in TMJ as early as 3 months post TM treatments, including decreased number of chondrocytes in superficial, middle, and deep layers with less safranin O staining in these areas (Fig. 2a, red arrows) and increased amounts of hypertrophic chondrocytes in superficial and middle layers (Fig. 2a, green arrows). At 4 months after TM injections, severe OA lesions, including spontaneous surface fissures at the superficial and middle layers (Fig. 2b, black arrowheads), loss of safranin O staining (Fig. 2b, red arrows), appearance of numerous disorganized rounded chondrocytes at superficial and deep layers (Fig. 2b, green arrows), and massive ectopic cartilage formation followed by new bone formation in mandibular condyle (Fig. 2b, blue arrows), were observed in cKO mice. Quantitative analyses revealed significantly higher Osteoarthritis Research Society International (OARSI) scores in cKO TMJs as compared with those in control TMJs (Fig. 2c) (P < 0.01, Student’s t-test). In addition, the safranin O-positive-stained cartilages in TMJs were decreased at 3 months, but markedly increased at 4 months, after TM injections (Fig. 2d). Subchondral bone damage and ectopic bone formation in TMJ were observed in cKO group at 4 months post TM treatments, as demonstrated by micro-computerized tomography (μCT) analyses (Fig. 3a). Moreover, quantitative μCT data showed that, when compared to control mice, the cKO mice displayed significantly decreased bone mineral density (BMD) (Fig. 3b), bone volume/tissue volume (BV/TV) (Fig. 3c) and trabecular thickness (Tb.Th) (Fig. 3d) and increased trabecular separation (Tb.Sp) (Fig. 3e) with no markedly altered trabecular number (Tb.N) (Fig. 3f). Taken together, the above findings demonstrate that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes results in severe osteoarthritic lesions in TMJ in adult mice.

Fig. 2
figure 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
figure 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. bf 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

Kindlin-2 loss reduces the expression of anabolic ECM proteins in condylar cartilages

We found that the expression levels of anabolic ECM proteins, including aggrecan, Col2a1 and Prg4, were all dramatically reduced in condylar cartilages of cKO mice, as demonstrated by IF staining analyses. (Fig. 4a). Quantitative analyses showed that the percentages of aggrecan-, Col2a1- and Prg4-positive cells were decreased by 24.75%, 42.5% and 45.75%, respectively, in cKO condylar cartilages vs. control condylar cartilages (Fig. 4b–d) (P < 0.001, cKO vs. control for all indicated parameters, Student’ t-test).

Fig. 4
figure 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. bg 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

Kindlin-2 deficiency promotes condylar chondrocyte hypertrophy and ECM degradation in condylar cartilages

During the development of OA, abnormal expression of Runx2 was reported to enhance chondrocyte hypertrophic differentiation, upregulate the expression of chondrocyte hypertrophic marker Col10a1, and lead to excessive production of ECM-degrading enzymes, such as matrix metalloproteinase (Mmp13).35,36 We found that expression of Runx2, Col10a1 and Mmp13 was extremely low in superficial and middle layers of condylar cartilages in control mice (Fig. 4a), which were all significantly upregulated in these areas in cKO relative to control mice (Fig. 4a, e–g) (P < 0.001, cKO group vs. control group for all quantitative parameters, Student’ t-test). Collectively, these data suggest that genetic ablation of Kindlin-2 in condylar chondrocytes enhances the hypertrophic differentiation of condylar chondrocytes and ECM degradation in TMJ cartilage.

Kindlin-2 loss inhibits condylar chondrocyte proliferation in TMJ

We further performed IF staining of cell proliferation marker Ki67 to assess whether the proliferation activity of condylar chondrocytes is affected by Kindlin-2 deletion. In control TMJs, Ki67 was highly expressed in condylar chondrocytes (Fig. 5a). However, the numbers of Ki67-positive chondrocytes were decreased by 25.2% in the superficial and middle layers of condylar cartilages in cKO TMJs compared to those in control TMJs (Fig. 5b) (P < 0.001, cKO vs. control, Student’ t-test).

Fig. 5
figure 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. bg 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

Kindlin-2 loss accelerates cell apoptosis in condylar chondrocytes in TMJ and in cultured ATDC5 cells

At 3 months after TM injection, Kindlin-2 loss significantly increased condylar chondrocyte apoptosis, as demonstrated by the terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) staining of TMJ sections of the two groups (Fig. 5a, c). Furthermore, expression of cleaved caspase 3, a critical executioner of intrinsic apoptosis, was significantly upregulated in condylar chondrocytes of cKO vs. control mice (Fig. 5a, d) (7.37% ± 2.50% in control group vs. 34.12% ± 5.98% in cKO group, P < 0.001, Student’s t-test). In contrast, the expression levels of p-Ampk, p-Akt and p-Erk were all markedly decreased in condylar chondrocytes of cKO mice compared with those in control mice (Fig. 5a, e–g), revealing a dysregulation of signaling pathways involved in cartilage homeostasis and survival caused by Kindlin-2 loss. To explore the underlying mechanism, we knocked down Kindlin-2 expression in ATDC5 chondrogenic cells by siRNA and found that it dramatically reduced the protein level of mitochondrial cytochrome c, a critical factor that is involved in mitochondrial apoptosis,37 and concomitantly increased the expression of cytosolic cytochrome c (Fig. 5h). In support of an increase in cell apoptosis, Kindlin-2 knockdown by siRNA significantly increased the levels of both total and cleaved caspases 3 proteins in ATDC5 cells (Fig. 5h). These findings demonstrate that loss of Kindlin-2 expression stimulates chondrocyte apoptosis in vitro and in TMJ.

Overexpression of Kindlin-2 enhances the production of ECM proteins and cell proliferation in ATDC5 cells

Next, we investigated whether overexpression of Kindlin-2 affects the expression of ECM proteins and cell proliferation in ATDC5 cells. Our data showed that overexpression of Kindlin-2 markedly enhanced the production of ECM proteins, such as Prg4 and Aggrecan, in ATDC5 cells (Fig. 5i). Moreover, we found that Kindlin-2 overexpression significantly enhanced the cell proliferation rate of ATDC5 cells (Fig. 5j).

Reduced Kindlin-2 expression in TMJ chondrocytes in aged mice

Aging is the most important independent risk factor for developing OA.38 We next determined the expression level of Kindlin-2 in TMJs in aged mice. TMJ samples were collected from healthy adult (4 months) or aged (18–22 months) mice and subjected to histological and IF staining. As expected, aged mice display multiple OA lesions in TMJs, including severe cartilage damage, surface fissures, ectopic cartilage formation, and subchondral bone sclerosis (Fig. 6a). Furthermore, the OARSI scores and safranin O-positive areas were higher in aged TMJs as compared with those in healthy adult TMJs (Fig. 6b, c). Importantly, we found that the protein level of Kindlin-2 was significantly lower in aged TMJs than in healthy adult TMJs, as demonstrated by IF staining analyses (Fig. 6d).

Fig. 6
figure 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


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