Osteoarthritis
and progression. 12–14 Because chondrocytes are the only cell type in articular cartilage, when they display dysfunctional metabolism, this leads to cartilage damage, which has been widely studied. Accumulated evidence shows that senescent chondrocytes are increased in human aged and OA cartilage lesions compared with that of young and healthy cartilage, suggesting a strong correlation between chondrocyte senes- cence and OA severity. 15 Senescent chondrocytes exhibit a senescence-associated secretory phenotype (SASP) and secrete enzymes capable of digesting cartilage extracellular matrix, resulting in cartilage degeneration and disruption. 16 17 Inter- estingly, senescent cell clearance in the mouse joint not only prevents disease progression but also maintains tissue struc- ture. 18 19 Together, chondrocyte senescence and cartilage ageing play essential roles in OA development; however, the factors that stimulate chondrocyte senescence and the under- lying mechanisms remain to be identified. As mechanosensitive cells, chondrocytes perceive and respond to mechanical stress throughout life. 20 21 Indeed, different types of exercise will lead to different stress inten- sities on cartilage, resting (0%–10%), walking and exercise (20%–40%), and joint trauma and injurious loading (60%– 90%). 22 23 Accumulating evidence indicates that high intensity mechanical stress accelerates chondrocyte catabolism, while moderate intensity stimulates the chondrocyte to secrete collagen. Moreover, high intensity stress can induce the chon- drocyte to express SASPs, and shear stress alone can induce chondrocyte senescence. 24–27 Therefore, chondrocyte senes- cence was identified as a turning point regarding chondrocyte phenotype in OA and its SASP activity is essential in cartilage erosion. However, the role of mechanical stress in chondro- cyte senescence and cartilage ageing is unclear and their mech- anistic association has not been reported. In this study, we found that mechanical overloading stim- ulated senescence in cultured chondrocytes and in mice articular cartilage, and identified that F-box and WD repeat domain containing 7 (FBXW7), a ubiquitin ligase, to be a key factor in the association between mechanical stress and chondrocyte senescence in OA pathology. FBXW7, as a ubiq- uitin ligase, is emerging as having a key role in controlling cell growth, differentiation and tumorigenesis, but its role in OA progression has not previously been investigated. Exces- sive mechanical loading downregulates FBXW7 to activate MKK7–JNK signalling, which stimulates chondrocyte senes- cence and consequently initiates and accelerates OA develop- ment. Targeting FBXW7-MKK7–JNK signalling represents a novel therapeutic approach for OA treatment. To examine the effect of mechanical loading on chondrocyte senescence, mouse primary chondrocytes were treated with 0.5 Hz and 5%, 10% and 20% cyclic tensile strain loading for 0–24 hours. Consistent with previous results, 0.5 Hz with 5% and 10% cyclic tensile strain loading upregulated Col2a1 but downregulated Mmp13 mRNA levels, indicating the chondro- genic effect of low mechanical loading in chondrocytes. 28 29 However, excessive mechanical loading by 20% cyclic tensile strain not only promoted catabolic effects but also stimu- lated chondrocyte senescence (figure 1A,B). The number of cells with senescence-associated β -galactosidase (SA- β Gal) staining, a classical indicator of senescence, and γ H2AX, a RESULTS Excessive mechanical loading induces chondrocyte senescence in vitro and in mice
marker of DNA damage, were increased in a time-dependent manner after excessive mechanical loading (figure 1C,D, and online supplemental figure S1A). In addition, 20% cyclic tensile strain loading increased the mRNA levels of p16 ink4a , p21 , Gadd45 and Il-6 but decreased LaminB1 , while 5% and 10% loading had a protective effect against cell senescence (online supplemental figure S1B–D). Furthermore, human primary chondrocytes were used to confirm the effect of mechanical loading on chondrocyte metabolism in both cyclic tensile strain loading model and compression loading model (online supplemental figure S2A–D). Interestingly, exam- ination of chondrocyte monolayer features in response to several stretch amplitudes revealed gradual, time-dependent reorientation of filamentous actin (F-actin) to the stretch direction. Monolayer alignment was initiated at 6 hour and completed at 24 hours of continuous 20% cyclic tensile strain. In contrast, 5% and 10% stretch were insufficient to trigger alignment, even at longer time scales (figure 1E). In addition, cyclic 20% strain resulted in an enlarged nuclear area, a feature of senescent cells (figure 1E,F). These findings demonstrate that excessive mechanical loading induces senes- cence in primary cultured chondrocytes. We further assessed the effect of mechanical overloading on chondrocyte senescence in mouse articular cartilage. As expected, the application of multiple loading episodes at a peak load of 13.5 N for 14 days induced proteoglycan loss with significant fibrillation of the articular surface on mouse knee joints, whereas no significant changes were detected on low mechanical loading (9 N). The number of articular chondrocytes stained for p16 INK4a and p21 increased mark- edly, accompanied by the loss of cartilage structure by 13.5 N peak loads (figure 1G,H). Together, these findings demon- strate that excessive mechanical loading accelerates chondro- cyte senescence in vitro and in articular cartilage, suggesting a potential mechanism in OA pathogenesis and development. Chondrocyte FBXW7 is reduced by mechanical overloading and is decreased in articular cartilage of patients with OA, aged mice and OA mice We subsequently investigated the mechanism through which mechanical overloading stimulates chondrocyte senescence. Isobaric tags for relative and absolute quantitation proteomic analysis was performed to quantitatively analyse and map proteins in mouse primary chondrocytes subjected to 20% elon- gation strain loading for 24 hours. Among the 813 differentially expressed proteins, FBXW7 was the most highly downregulated by mechanical stress (online supplemental table 1). FBXW7, a ubiquitin ligase and a member of the F-box family proteins, was of particular interest. It contributes to the degradation of proteins that positively regulate the cell cycle, but its role in chondrocyte and OA development is unknown. Immunohistochemical (IHC) staining of cartilage confirmed a marked decrease of chondrocyte FBXW7 by mechanical stress (figure 2A). Consistent with this, the Fbxw7 mRNA and protein levels were decreased by 20% cyclic tensile strain loading in human and mouse primary chon- drocyte culture (online supplemental figure S3A–C). Further- more, human primary chondrocytes were used to confirm the effect of mechanical loading on Fbxw7 expression in a compression loading model (online supplemental figure S3D). To further understand whether chondrocytes are responding to mechanical overload by producing less FBXW7 or are degrading FBXW7 protein more quickly, mouse primary chondrocytes were treated with MG132 (10 µ M) to inhibit proteolysis or with
Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513
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