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Kindlin-2 preserves integrity of the articular cartilage to protect against osteoarthritis

Nature Aging volume 2, pages 332–347 (2022)Cite this article

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Abstract

Osteoarthritis (OA) is an aging-related degenerative joint disease with a poorly defined mechanism. Here we report that kindlin-2 is highly expressed in articular chondrocytes and downregulated in the degenerated cartilage of aged mice and patients with OA. Kindlin-2 deletion in articular chondrocytes leads to spontaneous OA and exacerbates instability-induced OA lesions in adult mice. Kindlin-2 deficiency promotes mitochondrial oxidative stress and activates Stat3, leading to Runx2-mediated chondrocyte catabolism. Pharmacological inhibition of Stat3 activation or genetic ablation of Stat3 in chondrocytes reverses aberrant accumulation of Runx2 and extracellular-matrix-degrading enzymes and limits OA deteriorations caused by kindlin-2 deficiency. Deleting Runx2 in chondrocytes reverses structural changes and OA lesions caused by kindlin-2 deletion without downregulating p-Stat3. Intra-articular injection of AAV5–kindlin-2 decelerates progression of aging- and instability-induced knee joint OA in mice. Collectively, we identify a pathway consisting of kindlin-2, Stat3 and Runx2 in articular chondrocytes that is responsible for maintaining articular cartilage integrity and define a potential therapeutic target for OA.

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Fig. 1: Inducible deletion of kindlin-2 in chondrocytes causes striking spontaneous OA in adult mice.
Fig. 2: Kindlin-2 deficiency in chondrocytes accelerates instability-induced OA.
Fig. 3: Kindlin-2 loss induces chondrocyte hypertrophic differentiation and catabolism through Stat3-dependent upregulation of Runx2.
Fig. 4: Kindlin-2 deficiency promotes oxidative stress, Stat3 activation and nuclear translocation in chondrocytes.
Fig. 5: Kindlin-2 expression is drastically reduced in human OA articular cartilages.
Fig. 6: Genetic deletion of Stat3 in chondrocyte corrects Runx2 accumulation and attenuates OA lesions in cKO mice.
Fig. 7: Genetical deletion of Runx2 in chondrocytes palliates spontaneous and DMM-induced OA defects in cKO mice.
Fig. 8: Intra-articular injection of AAV5–kindlin-2 protects against development of aging- and DMM-induced OA in mice.

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Data availability

All data supporting the findings of this study are available within the article and its Extended Data files or from the corresponding authors upon reasonable request. Source data are provided with this paper. RNA-seq data have been deposited in Sequence Read Archive under accession no. PRJNA773746.

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Acknowledgements

The authors acknowledge the assistance of Core Research Facilities of Southern University of Science and Technology. This work was supported, in part, by the National Key Research and Development Program of China grant (2019YFA0906004), the National Natural Science Foundation of China grants (81991513, 82022047, 81630066, 81870532 and 82972100), the Guangdong Provincial Science and Technology Innovation Council grant (2017B030301018) and the Science and Technology Innovation Commission of Shenzhen Municipal Government grants (JCYJ20180302174246105, JCYJ20180302174117738 and KQJSCX20180319114434843). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Xiaohao Wu, Yumei Lai, Sheng Chen.

Authors and Affiliations

  1. Department of Biochemistry, School of Medicine, Southern University of Science and Technology, Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research Shenzhen Key Laboratory of Cell Microenvironment, Shenzhen, China

    Xiaohao Wu, Chu Tao, Xuekun Fu, Huiling Cao & Guozhi Xiao

  2. Department of Orthopedic Surgery, Rush University Medical Center, Chicago, IL, USA

    Yumei Lai & Jun Li

  3. Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Sheng Chen, Wei Tong, Hongtao Tian & Zengwu Shao

  4. Department of Medical Laboratory, Tianjin First Center Hospital, Tianjin Medical University, Tianjin, China

    Chunlei Zhou

  5. Department of Orthopedic Surgery, New York University School of Medicine, New York, NY, USA

    Chuanju Liu

  6. Department of Cell Biology, New York University School of Medicine, New York, NY, USA

    Chuanju Liu

  7. Research Center for Human Tissues and Organs Degeneration, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Di Chen

  8. Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, Department of Cell Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China

    Xiaochun Bai

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Contributions

G.X., X.W., Y.L. and H.C. were responsible for study design. X.W., Y.L., S.C., C.Z., X.F., C.T., J.L., W.T., H.T., X.B. and G.X. were responsible for study conduct and data collection. X.W., Y.L. and G.X. analyzed data. G.X., X.W., C.L., Z.S., X.B. and D.C. interpreted data. G.X. and X.W. drafted the manuscript. X.W., H.C. and G.X. are responsible for the integrity of data analysis.

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Correspondence to Xiaochun Bai, Huiling Cao or Guozhi Xiao.

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Extended data

Extended Data Fig. 1 K2 is highly expressed in mouse articular cartilage.

(a-b) Safranin O & Fast Green (SO&FG) and immunofluorescent (IF) staining of serial sections of mouse knee joint cartilage (a). Green double-headed arrow indicates hyaline cartilage; Red double-headed arrow indicates the calcified cartilage. White dashed lines indicate the cartilage surfaces. Red dashed line indicates the tide mark. Scale bar: 50 μm. Quantification of Kindlin-1-, 2- and -3-positive cells in articular cartilage (b). n = 4 biologically independent animals per group and results from one representative replicate are shown in a. Quantitative data are shown as mean ± standard deviation in b. The exact p values are shown. One-way ANOVA with Tukey’s multiple comparisons was used for statistical analysis.

Source data

Extended Data Fig. 2 Generation and characterizations of K2 cKO mice.

(a) Breeding strategy. (b) PCR genotyping using tail DNA. K2flox KO, ~300bp; K2flox WT, ~200bp; AggrecanCreERT2, ~650bp. (c) Representative images of SO&FG-stained and fluorescent images showing the tdTomato expression in knee joint sections from AggrecanCreERT2; Ai9 mice at 4 weeks after tamoxifen injections. S: synovium; C: cartilage. White dashed lines indicate the cartilage surfaces. Scale bar: 50 μm. (d, e) IF staining for expression of K2 in control and cKO synovium (d). Scale bar: 50 μm. Percentage of K2-positive cells in control and cKO synovium (e). n = 8 mice per group. (f) μCT scans of knee joints. Scale bar, 1.0 mm. n = 6 mice per group. (g) Representative images of Alcian blue/hematoxylin and eosin-stained knee sections. Higher magnification images of the boxed areas (right panels) are shown. Scale bar: 400 μm. n = 6 mice per group. (h-i) OARSI score. n = 6 mice per group. (j, k) Immunohistochemical (IHC) staining. Scale bar: 40 μm. n = 9 mice per group. (l-o) IF staining. Scale bar: 40 μm. n = 10 mice per group. Experiments in b and c were repeated at least three times independently with similar results. Quantitative data are shown as mean ± standard deviation in e, h, i, k, m and o. The exact p values are shown in figures. Two-sided unpaired t-test was used for statistical analysis.

Source data

Extended Data Fig. 3 Effects of K2 deletion in mouse primary articular chondrocytes.

(a) Isolation of primary articular chondrocytes from adult K2fl/fl; AggrecanCreERT2 male mice. (b-e) Western blotting analyses of expression of K2, p-Stat3/t-Stat3 and Runx2 in primary articular chondrocytes. Primary articular chondrocytes were isolated from 2-month-old K2fl/fl; AggrecanCreERT2 male mice and cultured in 6-well plates. The cells were then treated with 4-hydroxytamoxifen (cKO) and vehicle (control) for 48h. n = 3 biologically independent experiments for each group. (f) IF staining. Scale bar: 40 μm. (g) Representative images of primary articular chondrocytes attachment and spreading on type II collagen-coated surfaces at 6 and 24 h after seeding. Higher magnification images are shown in red dashed boxes. Scale bar: 50 μm. Primary articular chondrocytes were isolated from control and K2 cKO mice at 8 weeks after tamoxifen injections. Experiments in a, b, f and g were repeated at least three times independently with similar results. Quantitative data are shown as mean ± standard deviation in c, d and e. The exact p values are shown in figures. Two-sided unpaired t-test was used for statistical analysis.

Source data

Extended Data Fig. 4 siRNA knockdown / overexpression of Stat3 and Runx2 in chondrocytes.

(a) Quantitative data for Figure 3e. (b, c) siRNA knockdown of Runx2 and Stat3 in ATDC5 cells. siRNA #1380 (for Runx2) and #1962 (for Stat3) were used for subsequent experiments. (d) Quantitative data for Figure 3r. (e) Quantitative data for Figure 3s. (f) ATDC5 cells were transfected with si-NC or si-Stat3, followed by western blotting for expression of Stat3 and Runx2. (g) ATDC5 cells were transfected with empty vector (EV) and Stat3 expression vector (Stat3), followed by western blotting for expression of t-Stat3, p-Stat3 and Runx2. All experiments were repeated at least three times independently with similar results. Quantitative data are shown as mean ± standard deviation. The exact p values are shown in figures. In a, two-sided unpaired t-test was used for statistical analysis. In d and e, one-way ANOVA with Tukey’s multiple comparisons was used for statistical analysis.

Source data

Extended Data Fig. 5 Immunofluorescent analysis of articular chondrocytes in K2 cKO mice.

(a) IF staining for expression of K2, 9EG7, p-p38, p-Erk and p-Jak2 in articular chondrocytes from control and cKO mice at 6 months after tamoxifen injections. White dashed lines indicate the cartilage areas. Scale bar: 50 μm. (b-d) Percentage of positive-stained cells in control and cKO articular chondrocytes. For all experiments, n = 6 biologically independent animals per group and results from one representative replicate are shown in a. Quantitative data are shown as mean ± standard deviation in b-d. The exact p values are shown in figures. Two-sided unpaired t-test was used for statistical analysis.

Source data

Extended Data Fig. 6 Systemic inhibition of Stat3 activation limits OA lesions caused by K2 loss.

(a) A schematic diagram illustrating the experimental design. (b) Stattic improves restriction of movement of knee joint caused by K2 loss in DMM mice. (c) SO&FG (left panels), IF (middle panels) and H&E (right panels) staining of knee joint sections from cKO mice treated with PBS or Stattic for 2 months after DMM surgery. Scale bar, 50 μm. (d-g) OARSI score (d), cartilage area (e), osteophyte score (f) and synovitis score (g) were analyzed using histological sections. For all experiments, n = 8 biologically independent animals per group and results from one representative replicate are shown in b, c. Quantitative data are shown as mean ± standard deviation in d-g. The exact p values are shown in figures. Two-sided unpaired t-test was used for statistical analysis.

Source data

Extended Data Fig. 7 Breeding strategy and PCR genotyping.

(a) Breeding strategy for generating the K2fl/fl; Stat3fl/fl; AggrecanCreERT2 (KSdKO) mice. (b) PCR genotyping using tail DNA. K2flox KO, ~300bp; K2flox WT, ~200bp; Stat3flox KO, 187bp; Stat3flox WT, 146bp; AggrecanCreERT2, ~650bp. Primer sets are listed in Supplementary Table 4. Experiments in b were repeated at least three times independently with similar results.

Extended Data Fig. 8 Effect of Stat3 haploinsufficiency in chondrocytes on cartilage loss caused by K2 loss.

(a) A schematic diagram illustrating the experimental design. (b) SO&FG-stained sections knee joints of K2fl/fl; AggrecanCreERT2 mice and K2fl/fl; Stat3fl/+; AggrecanCreERT2 mice subjected to sham or DMM surgeries and tamoxifen injections as described in (a). Scale bar: 50 μm. (c) Quantification of cartilage loss. n = 8 biologically independent animals per group and results from one representative replicate are shown in b. Quantitative data are shown as mean ± standard deviation in c. The exact p values are shown in figures. Two-sided unpaired t-test was used for statistical analysis.

Source data

Extended Data Fig. 9 Effect of Runx2 haploinsufficiency in chondrocytes on cartilage loss caused by K2 loss.

(a) A schematic diagram illustrating the experimental design. (b) Alcian blue & orange G-stained sections of knee joints of K2fl/fl; AggrecanCreERT2 mice and K2fl/fl; Runx2fl/+; AggrecanCreERT2 mice subjected to sham or DMM surgeries and tamoxifen injections as described in (a). Scale bar: 50 μm. (c) Quantification of cartilage loss. n = 6 biologically independent animals per group and results from one representative replicate are shown in b. Quantitative data are shown as mean ± standard deviation in c. The exact p values are shown in figures. Two-sided unpaired t-test was used for statistical analysis.

Source data

Extended Data Fig. 10 Effect of Stat3/Runx2 double haploinsufficiency in chondrocytes on DMM-induced OA lesions.

(a) A schematic diagram illustrating the experimental design. (b) SO&FG-stained sections of the knee joints of Stat3fl/+; AggrecanCreERT2, Runx2fl/+; AggrecanCreERT2 and Stat3fl/+; Runx2fl/+; AggrecanCreERT2 mice subjected to DMM/sham surgery and tamoxifen (TAM)/corn oil injections as described in (a). Scale bar: 50 μm. (c) OARSI score. (d) Loss of cartilage. For all experiments, n = 6 biologically independent animals per group and results from one representative replicate are shown in b. Quantitative data are shown as mean ± standard deviation in c, d. The exact p values are shown in figures. Two-sided unpaired t-test was used for statistical analysis.

Source data

Supplementary information

Source data

Source Data Fig. 1

Statistical source data for Fig. 1.

Source Data Fig. 2

Statistical source data for Fig. 2.

Source Data Fig. 3

Statistical source data for Fig. 3.

Source Data Fig. 3

Unprocessed western blots for Fig. 3.

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Wu, X., Lai, Y., Chen, S. et al. Kindlin-2 preserves integrity of the articular cartilage to protect against osteoarthritis. Nat Aging 2, 332–347 (2022). https://doi.org/10.1038/s43587-021-00165-w

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