Recommended reads for EULAR 2024

Rheumatology at BMJ

Recommended reads for EULAR 2024

Contents Annals of the Rheumatic Diseases: Mechanical overloading promotes

chondrocyte senescence and osteoarthritis development through downregulating FBXW7 Annals of the Rheumatic Diseases: Tocilizumab in patients with new onset polymyalgia rheumatica (PMR-SPARE): a phase 2/3 randomised controlled trial RMD Open: Immune responses to mRNA vaccines against SARS-CoV-2 in patients with immune-mediated inflammatory rheumatic diseases Lupus Science & Medicine: Mezagitamab in systemic lupus erythematosus: clinical and mechanistic findings of CD38 inhibition in an autoimmune disease BMJ Case Reports: First use of tofacitinib to treat an immune checkpoint inhibitor- induced arthritis BMJ Case Reports: Reactive arthritis after COVID-19

Osteoarthritis

TRANSLATIONAL SCIENCE Mechanical overloading promotes chondrocyte senescence and osteoarthritis development through downregulating FBXW7 Haiyan Zhang, 1,2 Yan Shao, 1,2 Zihao Yao, 1,2 Liangliang Liu, 1,2 Hongbo Zhang, 1,2 Jianbin Yin, 1,2 Haoyu Xie, 1,2 Kai Li, 1,2 Pinglin Lai, 1,2 Hua Zeng, 1,2 Guozhi Xiao ‍ ‍, 3 Chun Zeng, 1,2 Daozhang Cai ‍ ‍, 1,2 Xiaochun Bai ‍ ‍ 1,2

ABSTRACT Objectives To investigate the role of mechanical stress in cartilage ageing and identify the mechanistic association during osteoarthritis (OA) progression. Methods F-box and WD repeat domain containing 7 (FBXW7) ubiquitin ligase expression and chondrocyte senescence were examined in vitro, in experimental OA mice and in human OA cartilage. Mice with Fbxw7 knockout in chondrocytes were generated and adenovirus-expressing Fbxw7 (AAV-Fbxw7) was injected intra-articularly in mice. Destabilised medial meniscus surgery was performed to induce OA. Cartilage damage was measured using the Osteoarthritis Research Society International score and the changes in chondrocyte senescence were determined. mRNA sequencing was performed in articular cartilage from Fbxw7 knockout and control mice. Results Mechanical overloading accelerated senescence in cultured chondrocytes and in mice articular cartilage. FBXW7 was downregulated by mechanical overloading in primary chondrocytes and mice cartilage, and decreased in the cartilage of patients with OA, aged mice and OA mice. FBXW7 deletion in chondrocytes induced chondrocyte senescence and accelerated cartilage catabolism in mice, as manifested by an upregulation of p16 INK4A , p21 and Colx and downregulation of Col2a1 and ACAN, which resulted in the exacerbation of OA. By contrast, intra-articular injection of adenovirus expressing Fbxw7 alleviated OA in mice. Mechanistically, mechanical overloading decreased Fbxw7 mRNA transcription and FBXW7-mediated MKK7 degradation, which consequently stimulated JNK signalling. In particular, inhibition of JNK activity by DTP3, a MKK7 inhibitor, ameliorated chondrocyte senescence and cartilage degeneration Conclusions FBXW7 is a key factor in the association between mechanical overloading and chondrocyte senescence and cartilage ageing in the pathology of OA. INTRODUCTION Osteoarthritis (OA) is the most common global age-related and post-traumatic degenerative joint disorder, which will become the disease with the highest disability rate globally by 2030. 1 2 Although mechanical overloading and advancing age have been recognised as the two most important risk factors for developing OA, much of the aetiology remains unkown. 3–5

Handling editor Josef S Smolen

Key messages

► Additional supplemental material is published online only. To view, please visit the journal online (http://d​x.​doi.​ org/1​ 0.​1136/a​ nnrheumdis-​ 2021-2​ 21513). For numbered affiliations see end of article. Correspondence to Dr Xiaochun Bai, Professor Daozhang Cai and Dr Chun Zeng, The Third Affiliated Hospital of Southern Medical University, Guangzhou, China; b​ aixc15@​smu.e​ du.​cn, c​ dz@​smu.e​ du.​cn, z​ engdavid@​126.c​ om

What is already known about this subject? ► Mechanical overloading and chondrocyte senescence play essential roles in osteoarthritis (OA) development. ► Fbxw7 deletion leads to p16 INK4a and p19 elevation to facilitate the cell cycle and promote cell senescence. What does this study add? ► F-box and WD repeat domain containing 7 (FBXW7), a ubiquitin ligase, is a key factor in the association between mechanical stress and chondrocyte senescence in OA pathology. ► Excessive mechanical loading downregulates FBXW7 to activate MKK7–JNK signalling, which stimulates chondrocyte senescence and consequently initiates and accelerates OA development.

HZ, YS and ZY contributed equally.

► Inhibition of JNK activity ameliorated chondrocyte senescence and cartilage degeneration.

Received 14 September 2021 Accepted 27 December 2021 Published Online First 20 January 2022

How might this impact on clinical practice or future developments? ► This study suggests that targeting FBXW7-­

MKK7–JNK signalling may be a novel therapeutic approach for OA treatment.

Proper mechanical loading is essential for joint health, while mechanical overloading can result in articular cartilage being prone to degenerative lesions that lead to OA onset and progression. Due to the progressive loss of articular carti- lage that mainly occurs in load-bearing joints, OA was previously, for many decades, consid- ered to be a mechanical issue. 6 In the clinic, the knee axis of most patients with OA is misaligned, resulting in various deformities leading to further exacerbation of wear and accelerating OA prog- ress. 7 8 Previous studies have shown that a variety of signalling pathways are activated during OA progression. However, the specific mechanism through which mechanical overloading induces OA has not been fully elucidated. 9–11 Advancing age has been identified as the prom- inent biological mechanism for OA development

© Author(s) (or their employer(s)) 2022. No

To cite: Zhang H, Shao Y, Yao Z, et al . Ann Rheum Dis 2022; 81 :676–686. commercial re-use. See rights and permissions. Published by BMJ.

Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513

676

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

677

Osteoarthritis

Figure 1 Excessive mechanical loading induces chondrocyte senescence in vitro and in mice. (A,B) Quantitative PCR analysis of Col2a1 and Mmp13 in primary chondrocytes treated with different elongation strain loading (5%,10% or 20%) for 0, 6, 12 and 24 hours. n=5 per time point. (C,D) Representative images and quantification of SA- β Gal staining in primary chondrocytes treated with different elongation strain loading (5%, 10% or 20%) for 0, 6, 12 and 24 hours. n=5 per time point. Scale bar: 50 µm. (E,F) Representative images and quantification of F-actin (phalloidin) staining in chondrocytes described in (C) . n=10 per time point. Scale bar: 50 µm. (G) Representative images of safranin O/Fast green staining (top) and immunofluorescence of p16 INK4a (middle) and p21 (bottom) in articular cartilage of mice treated with multiple loading episodes at peak loads of 9.0 and 13.5 N. Scale bars: 100 µm (first row) and 50 µm (second and third rows). (H) Quantitative analysis of the OARSI scale. n=5 per group. (I) Quantification of p16 INK4a -positive and p21-positive chondrocytes as a proportion of the total chondrocytes in mice cartilage described in (G). n=5 per group. Data are shown as mean±SD. Statistical analyses were conducted using one-way analysis of variance followed by Dunnett’s multiple comparison test (A,B,I), two-way analysis of variance followed by Dunnett’s multiple comparisons test (D,F) or Kruskal-Wallis test followed by Dunn’s multiple comparisons test (OARSI score) (H). Boxed area is enlarged in the bottom right corner. *P<0.05, **P<0.01. DAPI, 4',6-diamidino-2-­ phenylindole; NS, not significant; OARSI, Osteoarthritis Research Society International.

as previously reported. 30 These results indicate that the loss of FBXW7 protein at 24 hours under 20% elongation strain loading might be caused by the initial drop in the mRNA level at 6 hours (online supplemental figure S3G–J). Moreover, IF staining showed that FBXW7 was decreased in association with cartilage damage in patients with OA, which was further confirmed by western blotting analysis (figure 2B–E and online supplemental figure S4A–C). Inter- estingly, a loss of FBXW7-expressing chondrocytes was observed in aged (24-month-old) mice compared with young (4-month-old) mice (figure 2F–H and online supplemental figure S4D,E). Additionally, FBXW7-positive articular chon- drocytes were progressively reduced in a mechanical load-­ induced (destabilisation of the medial meniscus (DMM))

cycloheximide (CHX) (50 µ M) to block new protein synthesis under 20% elongation strain loading for 24 hours. When treated with CHX alone, the expression level of FBXW7 was reduced significantly. However, there was no significant difference in the levels of FBXW7 protein under mechanical overloading whether treated with CHX or not (online supplemental figure S3E). Furthermore, inhibition of proteolysis by MG132 increased FBXW7 protein level in control cells but could not prevent the marked decrease of FBXW7 induced by 20% elongation strain loading (online supplemental figure S3F). The results suggested that the loss of FBXW7 in response to mechanical overloading is mainly caused by a decrease in FBXW7 mRNA synthesis. In addition, when protein synthesis was blocked by CHX, we found that the level of FBXW7 decreased time-dependently

Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513

678

Osteoarthritis

Figure 2 Chondrocyte FBXW7 is reduced by mechanical overloading and decreased in OA articular cartilage. (A) IHC staining of FBXW7 in chondrocytes of mice treated with multiple loading episodes at peak loads of 13.5 N and controls, and quantitative analysis of FBXW7-positive chondrocytes as a proportion of the total chondrocytes. n=5 per group. Scale bar: 50 µm. P<0.001, 95% CI −39.30 to −20.16. (B) Representative images of safranin O/fast green staining (top) and immunofluorescence of FBXW7 (middle and bottom) in normal, moderate OA and severe OA cartilage. Scale bars: 200 µm (first row) and 100 µm (second row). (C,D) Quantitative analysis of OARSI scale and FBXW7-positive chondrocytes as a proportion of the total chondrocytes. n=5 per group. (E) Western blotting analysis of FBXW7 COL2A1, ADAMTS5, p16 INK4a and p21 in normal and severe OA cartilage. (F) Representative images of safranin O/fast green and IHC staining of FBXW7 in chondrocytes of mice aged 4 and 24 months. Scale bars: 100 µm (first row) and 50 µm (second row). (G) Quantification of the OARSI scale based on staining results in (F). n=10 per group. (H) Quantification of FBXW7-positive chondrocytes based on staining results in (F). n=10 per group. P<0.001, 95% CI −77.58 to −68.62. (I) Representative images of safranin O/fast green staining and immunofluorescence of FBXW7 in chondrocytes of controls and mice at 2, 4, 6 and 8 weeks after DMM surgery. Sale bars: 100 µm (first row) and 50 µm (second row). (J) Quantitative analysis of the OARSI scale of controls and DMM mice. n=10 per group. (K) Quantification of FBXW7-positive chondrocytes as a proportion of the total chondrocytes of control and DMM mice. n=10 per group. Data are shown as mean±SD. Statistical analyses were conducted using Student’s t-test (A,H), one-way analysis of variance followed by Tukey’s multiple comparison test (D,K), Kruskal-Wallis test followed by Dunn’s multiple comparisons test (OARSI score (C,J), or non-parametric Mann-Whitney U tests (OARSI score (G). Boxed area is enlarged in the bottom right corner. *P<0.05, **P<0.01. DMM, destabilisation of the medial meniscus; FBXW7, F-box and WD repeat domain containing 7; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IHC, immunohistochemical; NC, control; OA, osteoarthritis; OARSI, Osteoarthritis Research Society International.

S4F–H). Taken together, these observations show that chon- drocyte FBXW7 is reduced by mechanical overloading and is decreased in the articular cartilage of patients with OA, aged mice and OA mice, implicating a potential role for FBXW7 in

OA mouse model, which exhibited a senescence phenotype, indicating that both mRNA and protein levels of FBXW7 were reduced with the progression of articular cartilage degeneration (figure 2I–K and online supplemental figure

Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513

679

Osteoarthritis

24 hours. Increased expression of Col2a1 and decreased MMP13 were found in Ad-Fbxw7-treated chondrocytes, demonstrating that addition of FBXW7 can rescue the promotion of the cata- bolic effect caused by excessive mechanical loading (figure 4A,B). Subsequently, adenovirus expressing FBXW7 (AAV- Fbxw7 ) and comparable amounts of AAV-negative control were injected intra-­ articularly once a week from 3 days after DMM surgery. GFP distribution indicated that intra-articular injection of adenovirus mainly affected articular cartilage, accompanied by significantly elevated FBXW7 expression in the chondrocytes of the middle and deep zones in AAV- Fbxw7 -treated mice, demonstrating successful AAV-delivered overexpression of FBXW7 (figure 4C). As expected, AAV- Fbxw7 effectively alleviated OA development in mice, as manifested by reduced chondrocyte hypertrophic differentiation and attenuated cartilage destruction and proteo- glycan loss, together with increased Col2a1 expression and decreased MMP13 and Colx expression in the tibial cartilage of AAV- Fbxw7 -treated mice. Importantly, p16 INK4a -positive, p21-­ positive and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive articular chondrocytes were also significantly decreased by AAV- Fbxw7 (figure 4D, E and online supplemental figure S9A). Together, these data suggest that FBXW7 protects against mechanically induced chondrocyte senescence and OA development. To explore the mechanisms through which FBXW7 regu- lates chondrocyte senescence, the mRNA expression profile of articular cartilage from Fbxw7 KO mice and their littermate controls was analysed (SRA accession codes PRJNA78345). 31 By performing Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis, we found that genes related to the MAPK signalling pathway were abundantly expressed in Fbxw7 KO mouse cartilage, and MKK7 (Map2k7) was the most highly upregulated gene among those relating to the MAPK signalling pathway (figure 5A). It has been estab- lished that JNK activation can promote the ageing process. Phosphorylation of JNK did not significantly change in the articular cartilage of Fbxw7 KO mice at 3 months old but was markedly enhanced together with the protein level of MKK7, a MAPK kinase responsible for JNK activation, under stim- ulation of mechanical overload (figure 5B–D). By contrast, MKK4, another JNK signalling activator, remained unchanged in Fbxw7 KO cartilage (figure 5C). In addition, we further deci- phered the mechanism underlying the effects of FBXW7 on MKK7 and cartilage homeostasis using ATDC5 cells incubated with either Fbxw7 -overexpression adenovirus or siRNA. Results showed that FBXW7 associated with MKK7, and mediated MKK7 ubiquitination and degradation by proteasomes. Both the proteasome inhibitor MG132 and FBXW7 deficiency attenuated MKK7 ubiquitination and degradation (figure 5E,F). FBXW7 loss by excessive mechanical loading activates MKK7– JNK signalling to promote chondrocyte senescence We subsequently investigated whether the loss of FBXW7 promoted chondrocyte senescence via activation of MKK7. DTP3, a MKK7 inhibitor, inhibited JNK phosphorylation and rescued SA- β Gal staining enhanced by FBXW7 deletion in primary chondrocytes (figure 6A). Additionally, mechanical stress-stimulated γ H2AX, p16 ink4a , p21 , Gadd45 , Laminab1 , Mmp13 and Il-6 in chondrocytes from Fbxw7 KO mice could also be partially rescued by DTP3 (online supplemental figure S9B,C). Taken together, these results indicate that FBXW7 loss by excessive mechanical loading activates MKK7–JNK signalling to promote chondrocyte senescence.

the association between excessive mechanical load and chon- drocyte senescence during OA development.

FBXW7 deletion in chondrocytes accelerates cartilage ageing and exacerbates OA development in mice To determine the casual relationship between the loss of chon- drocyte FBXW7 and OA progression, we generated mice with a conditional deletion (knockout) of the Fbxw7 gene in chondro- cytes ( Fbxw7 KO) by crossing Fbxw7 flox/flox mice with Col2a1 -cre mice, and genotypes were determined by PCR (online supple- mental figure S5A,B). We found a slight decrease in the body length in Fbxw 7KO mice aged 4 weeks, and delayed formation of secondary ossification centre in Fbxw7 KO mice at postnatal day 7 (P7), compared with controls. However, no significant differences either in gross appearance or organisation of the articular cartilage and the growth plates were found between Fbxw7 KO mice and littermate controls at the ages of 4 and 12 weeks, together with no significant increased expression of COLX in growth plate. These results indicated that the retarded growth of Fbxw7 KO mice may cause by delayed formation of secondary ossification centre around P7 (online supplemental figures S5C–G and S6A–D). In addition, ablation of FBXW7 in articular chondrocytes of Fbxw 7KO mice was further confirmed by IHC staining (figure 3A). At the age of 3 months, no significant changes in chondro- cyte senescence markers were observed between control and Fbxw7 KO mice (online supplemental figure S6E). However, by the age of 18 months, Fbxw7 KO mice exhibited significant cartilage erosion and loss of both proteoglycans and cellularity in the articular cartilage compared with control mice, which was further validated by Osteoarthritis Research Society Inter- national (OARSI) scale analysis (figure 3B–D). The number of p16 INK4a - and p21-positive articular chondrocytes was mark- edly increased in aged Fbxw7 KO mice compared with their littermate controls. FBXW7 deletion also promoted chondro- cyte senescence in vitro because enhanced SA- β -galactosidase staining was observed in FBXW7-deficient primary chondrocyte culture at passage 6, indicating that the loss of FBXW7 induced chondrocyte senescence and contributed to OA development (figure 3B–D and online supplemental figure S7A). In addi- tion, FBXW7 deletion accelerated experimental OA in a DMM model. Colx and TUNEL-positive chondrocytes were increased, whereas Col2a1 and aggrecan were significantly decreased in Fbxw7 KO mice compared with control mice (figure 3E,F and online supplemental figure S7B,C). In addition, we also detected synovial inflammation and bone changes in Fbxw7 KO mice and their littermate controls. In both aged and DMM model mice, Fbxw7 KO mice developed larger periarticular osteophytes and more synovitis inflammation compared with littermate controls but exhibited no statistically significant difference in bone density (online supplemental figure S8A–D). These results indicate that the aggravated cartilage damage also accelerates synovial inflammation and osteophyte formation in Fbxw7 KO mice. Collectively, these results reveal that FBXW7 deletion in chondrocytes accelerates cell senescence and cartilage ageing, and leads to grossly observable cartilage destruction in aged and traumatic OA mice.

FBXW7 overexpression in cartilage alleviates OA development

We then performed an experiment with human or mouse primary chondrocytes treated with or without adenovirus containing FBXW7 (Ad- Fbxw7 ) under 20% elongation strain loading for

Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513

680

Osteoarthritis

Figure 3 Fbxw7 loss in chondrocyte accelerates mouse OA development. (A) Representative images of IHC staining of FBXW7 in articular cartilage of Fbxw7 KO and Control mice aged 3 months. Scale bar: 50 µm. (B) Representative images of H&E staining (first row), safranin O/fast green staining (second row) and IHC staining of p16 INK4a (third row) and p21 (fourth row) in articular cartilage of Fbxw7 KO and Control mice aged 18 months. Scale bars: 100 µm (first and second rows) and 50 µm (third and fourth rows). (C,D) Quantitative analysis of the OARSI scale and p16 INK4a -positive and p21-positive chondrocytes in Fbxw7 KO mice and Controls. n=10 per group. (E) Representative images of safranin O/fast green staining and immunofluorescence staining of Colx, COL2A1, aggrecan and TUNEL in chondrocytes of Fbxw7 KO and Controls at 4 and 8 weeks after DMM surgery. Scale bars: 100 µm (first row) and 50 µm (rest rows). (F) OARSI scale and quantification of Colx, ACAN and TUNEL-positive chondrocytes based on staining results in (E). n=10 per group. Data are shown as mean±SD. Statistical analyses were conducted using Student’s t-test (D), three-way analysis of variance followed by Tukey’s multiple comparison test (F) or non-parametric Mann-Whitney U tests (OARSI score) (C). Boxed area is enlarged in the bottom right corner. *P<0.05, **P<0.01. Con, control; DMM, destabilisation of the medial meniscus; FBXW7, F-box and WD repeat domain containing 7; IHC, immunohistochemical; NS, not significant; OA, osteoarthritis; OARSI, Osteoarthritis Research Society International; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling.

of DTP3 on OA were further verified in Fbxw7 KO mouse in which MKK7 was highly expressed in chondrocytes (figure 6C and online supplemental figure S10C,D). Taken together, these data suggest that FBXW7 loss promotes chondrocyte senescence and OA development partially through MKK7–JNK activation. DISCUSSION Ageing and mechanical overload play important roles in OA development. This study for the first time established a mech- anistic association between these two critical risk factors for

Inhibition of MKK7–JNK signalling delays chondrocyte senescence and OA development To further verify the role of MKK7 in OA development induced by mechanical overloading in vivo, DTP3 was injected intraperi- toneally to inhibit MKK7 after DMM surgery. The results showed a reduction in p-JNK-positive cells in articular cartilage by DTP3 injection and significant alleviation of the cartilage destruction and the OARSI score compared with the vehicle-treated mice. Catabolic factor expression and chondrocyte senescence and apoptosis were markedly reduced by DTP3 treatment (figure 6B and online supplemental figure S10A,B). These protective effects

Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513

681

Osteoarthritis

Figure 4 FBXW7 overexpression in chondrocytes alleviates experimental OA in mice. (A) Quantitative PCR analysis of COL2A1 and MMP13 in mouse primary chondrocytes treated with or without adenovirus containing FBXW7 (Ad- Fbxw7 ) under 20% elongation strain loading for 24 hours. n=6 per time point. (B) Quantitative PCR analysis of COL2A1 and MMP13 in human primary chondrocytes treated with or without Ad- Fbxw7 under 20% elongation strain loading for 24 hours. n=6 per time point. (C) Representative images and quantitative analysis of FBXW7-positive chondrocytes in the cartilage of mice intra-articularly injected with AAV-NC or AAV- Fbxw7 after DMM surgery. n=10 per group. Scale bar: 50 µm. (D) Safranin O/ fast green staining of joints from AAV- Fbxw7 and Control mice at 8 weeks after DMM surgery and quantitative analysis of the OARSI scale. n=10 per group. Scale bar: 100 µm. AAV-NC versus AAV-Fbxw7: p<0.002, 95% CI 0.5397 to 1.460. (E) Immunofluorescence staining and quantification of COL2A1, Colx, MMP13, p16 INK4a , p21 and TUNEL in chondrocytes of AAV- Fbxw7 and Control mice at 8 weeks after DMM surgery. n=10 per group. Scale bar: 50 µm. Data are shown as mean±SD. Statistical analyses were conducted using one-way analysis of variance followed by Tukey’s multiple comparison test (A–C,E), or Kruskal-Wallis test followed by Dunn’s multiple comparisons test (OARSI score (D). Boxed area is enlarged in the bottom right corner. *P<0.05, **P<0.01. AAV- Fbxw7 , adenovirus expressing FBXW7; AAV-NC, negative control; Con, control; DMM, destabilisation of the medial meniscus; FBXW7, F-box and WD repeat domain containing 7; NS, not significant, OA, osteoarthritis; OARSI, Osteoarthritis Research Society International; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling.

from patients undergoing total knee artificial implants has been noted, 32 but the direct relationship between the ageing process and OA development is not completely understood. 33 In the current study, we found senescent chondrocytes accumulated with age and in human OA cartilage. The presence of senescent chondrocytes near the osteoarthritic lesions, but not in intact tissue, further suggests an association between chondrocyte senescence and OA development. Additionally, mechanical over- load stimulated both primary chondrocytes and cartilage ageing in mice, indicating the interaction between biomechanics and the biological context during OA. FBXW7 has emerged as one of the substrate-recognition subunits of an SKP1-Cullin1-F-box protein (SCF)-type ubiquitin

OA. We showed that mechanical overloading stimulated chon- drocyte senescence in vitro and in mice articular cartilage, and identified FBXW7 as a key factor in the association between biomechanics and chondrocyte senescence in OA pathology. Excessive mechanical loading downregulated FBXW7 and thereby reduced FBXW7-mediated MKK7 ubiquitination and degradation. MKK7 accumulation subsequently activated JNK signalling, which promoted chondrocyte senescence and conse- quently accelerated cartilage degeneration and OA development. Supplementation of FBXW7 or inhibition of MKK7–JNK is thus a potential therapeutic target for OA treatment (figure 6D). Increasing age is strongly correlated with cartilage degener- ation and the presence of senescent cells in cartilage isolated

Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513

682

Osteoarthritis

Figure 5 FBXW7 regulates MKK7 ubiquitination and degradation. (A) Quantitative PCR analysis of Mkk7 in articular cartilage from Fbxw7 KO mice and littermate Controls. n=5 per group. P<0.002, 95% CI 1.471 to 1.867. (B) Quantitative PCR analysis of Mkk7 in primary chondrocytes from Fbxw7 KO mice and littermate Controls treated with or without tensile strain loading. n=5 per group. (C) Western blot and quantification of TAK1, MKK4, MKK7, p-JNK and JNK in mechanical stress-treated primary chondrocytes from Fbxw7 KO mice and Controls. (D) Representative images of immunofluorescence of p-JNK in cartilage from Fbxw7 KO mice and Controls, and quantitative analysis of p-JNK-positive chondrocytes compared with total chondrocytes. n=10 per group. Scale bar: 100 µm. (E) MKK7 was immunoprecipitated from ATDC5 cells after transfection with either adenovirus containing Fbxw7 (Ad- Fbxw7 ) or Fbxw7 -siRNA. The presence of MKK7 and FBXW7 in the immunoprecipitates was evaluated by immunoblotting. (F) MKK7 was immunoprecipitated from ATDC5 cells after stimulation with MG-132 and transfection with either Ad- Fbxw7 or Fbxw7 -siRNA. Western blotting detected the ubiquitination level of MKK7. Data are shown as mean±SD. Statistical analyses were conducted using Student’s t-test (A,D), two-way analysis of variance followed by Sidak’s multiple comparison test (B,C). Boxed area is enlarged in the bottom right corner. **P<0.01. Con, control; FBXW7, F-box and WD repeat domain containing 7; IB, immunoblotting; IP, immunoprecipitate; KO, knockout; NS, not significant.

Additionally, FBXW7 has been shown to negatively regulate mTOR signalling, which plays a vital role in OA development, as reported in our previous studies. 40–42 Although FBXW7 has been widely studied, its role in chondrocyte senescence and OA development has not been reported. An interesting finding of

(E3) ligase complex and targets several pathways for the degra- dation of various mammalian oncoproteins that control cell growth, differentiation, and tumorigenesis. 34–37 It has been reported that FBXW7 deletion leads to p16 INK4a and p19 eleva- tion to facilitate the cell cycle and promote cell senescence. 38 39

Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513

683

Osteoarthritis

Figure 6 Inhibition of MKK7–JNK signalling delays chondrocyte senescence and OA development. (A) Representative images and quantification of SA- β Gal staining and immunofluorescence staining of p-JNK in primary chondrocytes from Fbxw7 KO mice and controls after elongation strain loading for 24 hours with or without DTP3 treatment. n=5 per group. Scale bar: 50 µm. (B) Representative images and quantification of safranin O/fast green staining and immunofluorescence staining of p-JNK, p16 INK4a , p21 and TUNEL in chondrocytes of DTP3-treated mice (intraperitoneal injection, 10 mg/ kg, every other day) at 4 weeks post-DMM surgery. n=10 per group. Scale bars: 100 µm (first row) and 50 µm (the rest of the rows). (C) Safranin O/ fast green staining and immunofluorescence staining of p-JNK of joints from DTP3-treated Fbxw7 KO mice at 4 weeks post-DMM surgery. Quantitative analysis of the OARSI score and p-JNK-positive chondrocytes are shown on the right. n=5 per group. Scale bars: 100 µm (first row) and 50 µm (second row). (D) Schematic diagram representing molecular pathways in which excessive mechanical loading induces OA development through FBXW7. Data are shown as mean±SD. Statistical analyses were conducted using two-way analysis of variance followed by Sidak’s multiple comparison test (A), Student’s t-test (B and C), or non-parametric Mann-Whitney U tests (OARSI score) (B,C). Boxed area is enlarged in the bottom right corner. *P<0.05, **P<0.01. DMM, destabilisation of the medial meniscus; FBXW7, F-box and WD repeat domain containing 7; OA, osteoarthritis; OARSI, Osteoarthritis Research Society International; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling.

cartilage chondrocytes, which represents a novel mechanism for chondrocyte senescence during OA. To identify FBXW7 downstream signalling during chondro- cyte senescence and OA development, we screened out MKK7, and one MAPK kinase was shown to activate JNK. 43 MAPKs, including the JNK and p38MAPK signalling pathways, have been suggested to be extensively involved in OA. 44 JNK signalling can be phosphorylated by activating MKK4 or MKK7, while MKK4 can activate p38MAPK and JNK, whereas MKK7 is specifically involved in only JNK activation. 45–47 We found that MKK7, but not MKK4, participated in mechanical overloading-induced JNK activation. Although JNK signalling plays a key role in cell proliferation, differentiation and apoptosis in response to stress,

the present study was that FBXW7 was markedly downregu- lated in chondrocytes by excessive mechanical loading both in vitro and in vivo. FBXW7 deletion in chondrocytes resulted in the senescence and degeneration of articular cartilage and exac- erbation of OA. In addition, we detected senescent chondro- cytes in articular cartilage of patients with OA and DMM OA mice, which were enhanced by FBXW7 deletion and reversed by FBXW7 overexpression. However, no significant changes in chondrocyte senescence were observed between Fbxw7 KO mice and controls at a young age, indicating that FBXW7 defi- ciency alone was not sufficient to induce cell senescence, unless under the context of mechanical loading or old age. Therefore, we propose that mechanical overloading reduces FBXW7 in

Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513

684

Osteoarthritis

the contributions of MKK7–JNK in cartilage ageing and OA progression in response to mechanical stress are not known. 48 49 In the present study, we found that FBXW7 inhibition by exces- sive mechanical loading strongly elevated MKK7 expression and subsequently induced an increase in the JNK signal in chon- drocytes, resulting in enhanced cell senescence. Additionally, FBXW7 loss-induced chondrocyte senescence and cartilage degeneration were ameliorated by MKK7 inhibitor DTP3. These results indicate that the MKK7–JNK pathway plays an important role in mechanical stress-induced chondrocyte senescence and cartilage degeneration. To conclude, our study found an association between mechan- ical loading and cell senescence in OA development. FBXW7 loss and activation of MKK7–JNK signalling play a crucial role in biomechanically induced chondrocyte senescence. Overex- pression of FBXW7 by targeting its transcriptional regulators or upstream lncRNAs, or targeting MKK7 by DTP3 might repre- sent novel therapeutic approaches for OA treatment. Author affiliations 1 Department of Orthopedics, Academy of Orthopedics·Guangdong Province, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, The Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, China 2 Department of Joint Surgery, Center for Orthopedic Surgery, Orthopedic Hospital of Guangdong Province, The Third School of Clinical Medicine, Southern Medical University, The Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, China 3 Department of Biochemistry, School of Medicine, Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Shenzhen Key Laboratory of Cell Microenvironment, Southern University of Science and Technology, Shenzhen, Guangdong, China Contributors HyZ and XB conceived the ideas for experimental designs, analysed data and wrote the manuscript. HyZ and YS conducted the majority of the experiments and helped with manuscript preparation. ZY conducted the majority of the experiments and analysed data during the revision of the article. CZ, HbZ and LL performed immunohistochemistry and immunofluorescence and confocal imaging. HbZ and KL conducted cell cultures and western blot experiments. JY and ZY collected human tissue samples. CZ, XB and DC developed the concept, supervised the project and conceived the experiments. All authors approved the final version of the manuscript. XB accepted full responsibility for the finished work, had access to the data and controlled the decision to publish. Funding This work was supported by grants from the National Natural Science Foundation of China (grant numbers 81974341, 81991510 and 81991511) and the Natural Science Foundation of Guangdong Province (2020A1515011062). Competing interests None declared. Patient consent for publication Consent obtained directly from patient(s) Ethics approval This study involves human participants, and patient consent and the approval of the ethics committee of the Third Affiliated Hospital of Southern Medical University (Guangzhou, China) were obtained before the human tissue samples were harvested (201711001). Participants gave informed consent to participate in the study before taking part. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data are available in a public, open access repository. Not applicable. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. ORCID iDs

REFERENCES 1 Sharma L. Osteoarthritis of the knee. N Engl J Med 2021;384:51–9. 2 Johnson VL, Hunter DJ. The epidemiology of osteoarthritis. Best Pract Res Clin Rheumatol 2014;28:5–15. 3 Visser AW, de Mutsert R, le Cessie S, et al . The relative contribution of mechanical stress and systemic processes in different types of osteoarthritis: the Neo study. Ann Rheum Dis 2015;74:1842–7. 4 Murphy MP, Koepke LS, Lopez MT, et al . Articular cartilage regeneration by activated skeletal stem cells. Nat Med 2020;26:1583–92. 5 Jeon OH, David N, Campisi J, et al . Senescent cells and osteoarthritis: a painful connection. J Clin Invest 2018;128:1229–37. 6 Glyn-Jones S, Palmer AJR, Agricola R, et al . Osteoarthritis. Lancet 2015;386:376–87. 7 Cooke ME, Lawless BM, Jones SW, et al . Matrix degradation in osteoarthritis primes the superficial region of cartilage for mechanical damage. Acta Biomater 2018;78:320–8. 8 Pierson E, Cutler DM, Leskovec J, et al . An algorithmic approach to reducing unexplained pain disparities in underserved populations. Nat Med 2021;27:136–40. 9 Chang SH, Mori D, Kobayashi H, et al . Excessive mechanical loading promotes osteoarthritis through the gremlin-1-NF- κ B pathway. Nat Commun 2019;10:1442. 10 Richard D, Liu Z, Cao J, et al . Evolutionary selection and constraint on human knee chondrocyte regulation impacts osteoarthritis risk. Cell 2020;181:362–81. 11 Barenius B, Ponzer S, Shalabi A, et al . Increased risk of osteoarthritis after anterior cruciate ligament reconstruction: a 14-year follow-up study of a randomized controlled trial. Am J Sports Med 2014;42:1049–57. 12 Kelly PN. Targeting senescence to combat osteoarthritis. Science 2017;356:595.2–6. 13 Childs BG, Gluscevic M, Baker DJ, et al . Senescent cells: an emerging target for diseases of ageing. Nat Rev Drug Discov 2017;16:718–35. 14 Coryell PR, Diekman BO, Loeser RF. Mechanisms and therapeutic implications of cellular senescence in osteoarthritis. Nat Rev Rheumatol 2021;17:47–57. 15 Xu M, Bradley EW, Weivoda MM, et al . Transplanted senescent cells induce an Osteoarthritis-Like condition in mice. J Gerontol A Biol Sci Med Sci 2017;72:780–5. 16 He S, Sharpless NE. Senescence in health and disease. Cell 2017;169:1000–11. 17 Xie J, Wang Y, Lu L, et al . Cellular senescence in knee osteoarthritis: molecular mechanisms and therapeutic implications. Ageing Res Rev 2021;70:101413. 18 Jeon OH, Kim C, Laberge R-M, et al . Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nat Med 2017;23:775–81. 19 Batshon G, Elayyan J, Qiq O, et al . Serum NT/CT SIRT1 ratio reflects early osteoarthritis and chondrosenescence. Ann Rheum Dis 2020;79:1370–80. 20 Vincent TL, Wann AKT. Mechanoadaptation: articular cartilage through thick and thin. J Physiol 2019;597:1271–81. 21 Lee H-P, Gu L, Mooney DJ, et al . Mechanical confinement regulates cartilage matrix formation by chondrocytes. Nat Mater 2017;16:1243–51. 22 Miller RH. Joint loading in runners does not initiate knee osteoarthritis. Exerc Sport Sci Rev 2017;45:87–95. 23 Davis S, Roldo M, Blunn G, et al . Influence of the mechanical environment on the regeneration of osteochondral defects. Front Bioeng Biotechnol 2021;9:603408. 24 Zhen G, Guo Q, Li Y, et al . Mechanical stress determines the configuration of TGF β activation in articular cartilage. Nat Commun 2021;12:12. 25 Griffin TM, Guilak F. The role of mechanical loading in the onset and progression of osteoarthritis. Exerc Sport Sci Rev 2005;33:195–200. 26 Arokoski JP, Jurvelin JS, Väätäinen U, et al . Normal and pathological adaptations of articular cartilage to joint loading. Scand J Med Sci Sports 2000;10:186–98. 27 Buschmann MD, Hunziker EB, Kim YJ, et al . Altered aggrecan synthesis correlates with cell and nucleus structure in statically compressed cartilage. J Cell Sci 1996;109 (Pt 2:499–508. 28 Pingguan-Murphy B, Nawi I. Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading. Clinics 2012;67:939–44. 29 He Z, Leong DJ, Zhuo Z, et al . Strain-Induced mechanotransduction through primary cilia, extracellular ATP, purinergic calcium signaling, and ERK1/2 transactivates Cited2 and downregulates MMP-1 and MMP-13 gene expression in chondrocytes. Osteoarthritis Cartilage 2016;24:892–901. 30 Lan H, Tan M, Zhang Q, et al . Lsd1 destabilizes FBXW7 and abrogates FBXW7 functions independent of its demethylase activity. Proc Natl Acad Sci U S A 2019;116:12311–20. 31 Zhang H, Shao Y, Yao Z. Data from: mechanical overloading promotes chondrocyte senescence and osteoarthritis development through downregulating FBXW7. NCBI, 2021. Available: https://www.ncbi.nlm.nih.gov/sra/PRJNA783453 32 Martin JA, Buckwalter JA. Telomere erosion and senescence in human articular cartilage chondrocytes. J Gerontol A Biol Sci Med Sci 2001;56:B172–9. 33 McCulloch K, Litherland GJ, Rai TS. Cellular senescence in osteoarthritis pathology. Aging Cell 2017;16:210–8. 34 Yeh C-H, Bellon M, Nicot C. Fbxw7: a critical tumor suppressor of human cancers. Mol Cancer 2018;17:115. 35 He J, Song Y, Li G, et al . Fbxw7 increases CCL2/7 in CX3CR1hi macrophages to promote intestinal inflammation. J Clin Invest 2019;129:3877–93.

Guozhi Xiao http://orcid.org/0000-0002-4269-2450 Daozhang Cai http://orcid.org/0000-0002-8232-8194 Xiaochun Bai http://orcid.org/0000-0001-9631-4781

Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513

685

Osteoarthritis

36 Close V, Close W, Kugler SJ, et al . FBXW7 mutations reduce binding of NOTCH1, leading to cleaved NOTCH1 accumulation and target gene activation in CLL. Blood 2019;133:830–9. 37 Lan H, Sun Y. Fbxw7 E3 ubiquitin ligase: degrading, not degrading, or being degraded. Protein Cell 2019;10:861–3. 38 Cui D, Xiong X, Shu J, et al . Fbxw7 confers radiation survival by targeting p53 for degradation. Cell Rep 2020;30:497–509. 39 Wang L, Chen R, Li G, et al . Fbw7 mediates senescence and pulmonary fibrosis through telomere uncapping. Cell Metab 2020;32:860–77. 40 Mao J-H, Kim I-J, Wu D, et al . Fbxw7 targets mTOR for degradation and cooperates with PTEN in tumor suppression. Science 2008;321:1499–502. 41 Zhang H, Wang H, Zeng C, et al . Mtorc1 activation downregulates FGFR3 and PTH/ PTHrP receptor in articular chondrocytes to initiate osteoarthritis. Osteoarthritis Cartilage 2017;25:952–63. 42 Zhang H, Lin C, Zeng C, et al . Synovial macrophage M1 polarisation exacerbates experimental osteoarthritis partially through R-spondin-2. Ann Rheum Dis 2018;77:1524–34.

43 Kragelj J, Palencia A, Nanao MH, et al . Structure and dynamics of the MKK7-JNK signaling complex. Proc Natl Acad Sci U S A 2015;112:3409–14. 44 Zhou F, Mei J, Han X, et al . Kinsenoside attenuates osteoarthritis by repolarizing macrophages through inactivating NF- κ B/MAPK signaling and protecting chondrocytes. Acta Pharm Sin B 2019;9:973–85. 45 Vander Griend DJ, Kocherginsky M, Hickson JA, et al . Suppression of metastatic colonization by the context-dependent activation of the c-Jun NH2-terminal kinase kinases JNKK1/MKK4 and MKK7. Cancer Res 2005;65:10984–91. 46 Syc-Mazurek SB, Rausch RL, Fernandes KA, et al . Mkk4 and MKK7 are important for retinal development and axonal injury-induced retinal ganglion cell death. Cell Death Dis 2018;9:1095. 47 Haun F, Neumann S, Peintner L, et al . Identification of a novel anoikis signalling pathway using the fungal virulence factor gliotoxin. Nat Commun 2018;9:3524. 48 Wagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK pathways in cancer development. Nat Rev Cancer 2009;9:537–49. 49 Ugbode C, Garnham N, Fort-Aznar L, et al . Jnk signalling regulates antioxidant responses in neurons. Redox Biol 2020;37:101712.

Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513

686

Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 9 Page 10 Page 11 Page 12 Page 13 Page 14 Page 15 Page 16 Page 17 Page 18 Page 19 Page 20 Page 21 Page 22 Page 23 Page 24 Page 25 Page 26 Page 27 Page 28 Page 29 Page 30 Page 31 Page 32 Page 33 Page 34 Page 35 Page 36 Page 37 Page 38 Page 39 Page 40 Page 41 Page 42 Page 43 Page 44 Page 45 Page 46 Page 47

bmjgroup.com

Powered by FlippingBook