Objective: Autophagy is a process for turnover of intracellular organelles and molecules that protects cells during stress responses. We undertook this study to evaluate the potential roles of Unc‐51–like kinase 1 (ULK1), an inducer of autophagy, Beclin1, a regulator of autophagy, and microtubule‐associated protein 1 light chain 3 (LC3), which executes autophagy, in the development of osteoarthritis (OA) and in cartilage cell death.Methods: Expression of ULK1, Beclin1, and LC3 was analyzed in normal and OA human articular cartilage and in knee joints of mice with aging‐related and surgically induced OA, using immunohistochemistry and Western blotting. Poly(ADP‐ribose) polymerase (PARP) p85 expression was used to determine the correlation between cell death and autophagy.Results: ULK1, Beclin1, and LC3 were constitutively expressed in normal human articular cartilage. ULK1, Beclin1, and LC3 protein expression was reduced in OA chondrocytes and cartilage, but these 3 proteins were strongly expressed in the OA cell clusters. In mouse knee joints, loss of glycosaminoglycans (GAGs) was observed at ages 9 months and 12 months and in the surgical OA model, 8 weeks after knee destabilization. Expression of ULK1, Beclin1, and LC3 decreased together with GAG loss, while PARP p85 expression was increased.Conclusion: Autophagy may be a protective or homeostatic mechanism in normal cartilage. In contrast, human OA and aging‐related and surgically induced OA in mice are associated with a reduction and loss of ULK1, Beclin1, and LC3 expression and a related increase in apoptosis. These results suggest that compromised autophagy represents a novel mechanism in the development of OA.

Objectives Osteoarthritis is associated with cell death and extracellular matrix degradation in articular cartilage. Autophagy is an essential cellular homeostasis mechanism that was found to be deficient in ageing and osteoarthritic cartilage. This study determined whether pharmacological inhibition of the mammalian target of rapamycin (mTOR), a key inhibitor of autophagy, has disease-modifying activity in experimental osteoarthritis. Methods Experimental osteoarthritis was induced by transection of the medial meniscotibial ligament and the medial collateral ligament in 2-month-old C57Bl/6 mice (n=36). Rapamycin (1 mg/kg weight/day) (n=18 mice) or dimethyl sulphoxide vehicle control (n=18 mice) was administered intraperitoneally for 10 weeks. Histopathological changes in articular cartilage and synovium were examined by using semiquantitative scoring systems. Rapamycin effects on mTOR signalling, autophagy, cartilage homeostasis and inflammation were analysed by immunohistochemistry and immunofluorescence staining. Results Rapamycin affected the mTOR signalling pathway in mouse knee joints as indicated by the inhibition of ribosomal protein S6 phosphorylation, a target of mTOR and activation of LC3, a main marker of autophagy. The severity of cartilage degradation was significantly (p<0.01) reduced in the rapamycin-treated group compared with the control group and this was associated with a significant (p<0.05) decrease in synovitis. Rapamycin treatment also maintained cartilage cellularity and decreased ADAMTS-5 and interleukin-1β expression in articular cartilage. Conclusions These results suggest that rapamycin, at least in part by autophagy activation, reduces the severity of experimental osteoarthritis. Pharmacological activation of autophagy may be an effective therapeutic approach for osteoarthritis.

Osteoarthritis (OA) is the most common joint disease and typically begins with an aging-related disruption of the articular cartilage surface. Mechanisms leading to the aging-related cartilage surface degeneration remain to be determined. Here, we demonstrate that nonhistone chromatin protein high-mobility group box (HMGB) protein 2 is uniquely expressed in the superficial zone (SZ) of human articular cartilage. In human and murine cartilage, there is an aging-related loss of HMGB2 expression, ultimately leading to its complete absence. Mice genetically deficient in HMGB2 ( Hmgb2 ^−/− ) show earlier onset of and more severe OA. This is associated with a profound reduction in cartilage cellularity attributable to increased cell death. These cellular changes precede glycosaminoglycan depletion and progressive cartilage erosions. Chondrocytes from Hmgb2 ^−/− mice are more susceptible to apoptosis induction in vitro . In conclusion, HMGB2 is a transcriptional regulator specifically expressed in the SZ of human articular cartilage and supports chondrocyte survival. Aging is associated with a loss of HMGB2 expression and reduced cellularity, and this contributes to the development of OA.

Objective: A major signaling pathway that regulates cellular aging is the insulin/insulin‐like growth factor 1 (IGF‐1)/phosphatidylinositol 3‐kinase (PI3K)/Akt/FoxO transcription factor axis. We previously observed that FoxO transcription factors are dysregulated in aged and OA cartilage. The objective of this study was to investigate the impact of down‐regulated FoxO transcription factors on chondrocytes.Methods: Small interfering RNAs (siRNAs) targeting FOXO1 (siFOXO1) and FOXO3 (siFOXO3) were transfected into human articular chondrocytes. Cell viability following treatment with the oxidant tert‐butyl‐hydroperoxide (tBHP) was measured by MTT assay. Caspase 3/7 activation and apoptotic cells were examined. Gene and protein expression of antioxidant proteins and autophagy‐related proteins and changes in inflammatory mediators following treatment with interleukin‐1β were assessed. Cells transfected with FOXO plasmids were also analyzed.Results: Cell viability was significantly reduced by siFOXO after treatment with tBHP. Apoptosis accompanied by caspase activation was significantly increased in siFOXO‐transfected chondrocytes. Knockdown of FOXO1 and FOXO1+3 resulted in significant reductions in levels of glutathione peroxidase 1 (GPX‐1), catalase, light chain 3 (LC3), Beclin1, and sirtuin 1 (SIRT‐1) proteins following treatment with tBHP. In contrast, the constitutive active form of FOXO3 increased cell viability while inducing GPX‐1, Beclin1, and LC3 in response to tBHP. Expression and production of ADAMTS‐4 and chemerin were significantly increased in siFOXO‐transfected chondrocytes.Conclusion: Reduced expression of FoxO transcription factors in chondrocytes increased susceptibility to cell death induced by oxidative stress. This was associated with reduced levels of antioxidant proteins and autophagy‐related proteins. Our data provide evidence for a key role of FoxO transcription factors as regulators of chondrocyte oxidative stress resistance and tissue homeostasis.

Objective: Aging is a main risk factor for osteo arthritis (OA), the most prevalent musculoskeletal disorder. Defects in autophagy, an essential cellular homeostasis mechanism, have recently been observed in OA articular cartilage. The objectives of this study were to establish the constitutive level of autophagy activation in normal cartilage and to monitor the temporal relationship between changes in autophagy and aging‐related degradation of cartilage in a mouse model.Methods: In GFP‐LC3–transgenic mice, green fluo rescent protein (GFP)–light chain 3 (LC3) is ubiquitously expressed, and the accumulation of GFP puncta, repre senting autophagosomes, was quantified by confocal microscopy as a measure of autophagy activation. Expression of the autophagy proteins autophagy‐related protein 5 (ATG‐5) and microtubule‐associated protein 1 light chain 3 (LC3) was analyzed by immunohistochemistry. Cartilage cellularity, apoptotic cell death, and cartilage structural damage and changes in synovium and bone were examined by histology and immunohistochemistry.Results: Basal autophagy activation was detected in liver and knee articular cartilage from young (6‐month‐old) mice, with higher levels in cartilage than in liver in the same animals. In 28‐month‐old mice, there was a statistically significant reduction in the total number of autophagic vesicles per cell (P < 0.01) and in the total area of vesicles per cell (P < 0.01) in the articular cartilage as compared to that from young 6‐month‐old mice. With increasing age, the expression of ATG‐5 and LC3 decreased, and this was followed by a reduction in cartilage cellularity and an increase in the apoptosis marker poly(ADP‐ribose) polymerase p85. Cartilage structural damage progressed in an age‐dependent manner subsequent to the autophagy changes.Conclusion: Autophagy is constitutively activated in normal cartilage. This is compromised with aging and precedes cartilage cell death and structural damage.