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
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