Deletion of JNK Enhances Senescence in Joint Tissues and Increases the Severity of Age-Related Osteoarthritis in Mice

Abstract

Objective: To determine the role of JNK signaling in the development of osteoarthritis (OA) induced by joint injury or aging in mice.

Methods: In the joint injury model, 12-week-old wild-type control, JNK1^-/- , JNK2^-/- , and JNK1^fl/fl JNK2^-/- aggecan-Cre^{ERT 2} double-knockout mice were subjected to destabilization of the medial meniscus (DMM) (n = 15 mice per group) or sham surgery (n = 9-10 mice per group), and OA was evaluated 8 weeks later. In the aging experiment, wild-type control, JNK1^-/- , and JNK2^-/- mice (n = 15 per group) were evaluated at 18 months of age. Mouse knee joints were evaluated by scoring articular cartilage structure, toluidine blue staining, osteophytes, and synovial hyperplasia, by histomorphometric analysis, and by immunostaining for the senescence marker p16^{INK 4a} . Production of matrix metalloproteinase 13 (MMP-13) in cartilage explants in response to fibronectin fragments was measured by enzyme-linked immunosorbent assay.

Results: There were no differences after DMM surgery between the wild-type and the JNK-knockout mouse groups in articular cartilage structure, toluidine blue, or osteophyte scores or in MMP-13 production in explants. All 3 knockout mouse groups had increased subchondral bone thickness and area of cartilage necrosis compared to wild-type mice. Aged JNK-knockout mice had significantly worse articular cartilage structure scores compared to the aged wild-type control mice (mean ± SD 52 ± 24 in JNK1^-/- mice and 60 ± 25 in JNK2^-/- mice versus 32 ± 18 in controls; P = 0.02 and P = 0.004, respectively). JNK1^-/- mice also had higher osteophyte scores. Deletion of JNK resulted in increased expression of p16^{INK 4a} in the synovium and cartilage in older mice.

Conclusion: JNK1 and JNK2 are not required for the development of OA in the mouse DMM model. Deletion of JNK1 or JNK2 is associated with more severe age-related OA and increased cell senescence, suggesting that JNK may act as a negative regulator of senescence in the joint.

Figure 1.

Effects of JNK deletion on histologic measures of OA after destabilization of the medial meniscus (DMM). JNK1^−/−, JNK2^−/−, JNK1/2 dKO, and wild-type (WT) control mice underwent DMM or sham control surgery at 12 weeks of age. Eight weeks after surgery the mice were sacrificed and the operated joints evaluated histologically. A, Articular cartilage structure (ACS) scores performed on the medial (MTP) and lateral (LTP) tibial plateus from six sections per joint were summed. B, Toludine (Tol) blue staining was scored on the MTP and LTP using a mid-coronal section from each joint. C, Osteophytes were scored on the MTP and LTP from a mid-coronal section. D, Synovial hyperplasia was scored on the medial side of a mid-coronal section from each joint. Numbers of mice in each group are provided in Supplementary Table 1. Results shown are mean ± standard deviation.*p<0.0001, **p<0.001, ***p<0.01 by Mann-Whitney test comparing sham to DMM within each genotype and Kruskal Wallis test adjusted for multiple comparisons of WT DMM to each JNK knockout DMM group.

Figure 2.

Effects of JNK deletion on histologic measures of OA in 18 month-old mice. JNK1^−/−, JNK2^−/− and wild-type (WT) mice were evaluated. A, Articular cartilage structure (ACS) scores performed on the medial (MTP) and lateral (LTP) tibial plateus from six sections per joint were summed. B, Osteophytes were scored on the MTP and LTP from a mid-coronal section. Results shown are mean ± standard deviation and analysis by Mann-Whitney test comparing WT controls to each genotype.

Figure 3.

Fibronectin fragment (FN-f) stimulation of active MMP-13 in cultured explants from wild-type (WT) and JNK knockout mice. Explants harvested as femoral caps were stimulated in serum-free conditions with 1μM FN-f for 48 hours. Conditioned media was removed and assayed for active MMP-13 using an activity-based ELISA. Results shown are mean ± standard deviation. *p=0.005, **p=0.02, ***p<0.0001, †p<0.001 by paired t-test comparing control to FN-f stimulation for each genotype.

Figure 4.

Effects of JNK deletion on p16^Ink4a as a marker of cell senescence in joint tissues. A, Representative sections of immunohistochemical staining of patellofemoral sections from a wild-type and a JNK1^−/− mouse. 100x magnification. Insets 400x magnification. Brown immunostaining is seen in the synovial tissue of the JNK1^−/− but not the wild-type mouse. B, The number of cells positive for p16^Ink4a in the synovium were counted for n=9 mice per genotype. Results shown are mean±standard deviation and analysis by unpaired t-tests comparing the wild-type control to each genotype. C, Representative result of immunofluorescent staining for p16^Ink4a in a tibiofemoral section of a JNK1^−/− mouse. 100x magnification. Synovium, white arrowheads. D, Immunoblots of lysates obtained from JNK2^−/− and wild-type (wt) mouse cartilage probed with an antibody for p16^Ink4a, JNK2 and GAPDH as a loading control. Cell lysates from mouse embryonic fibroblasts (MEFs) expressing p16 (MEF2) or not expressing p16 (MEF6) were used as positive and negative controls.