Objective Autophagy, is a key pathway of cellular homeostasis for removing damaged macromolecules and organelles, including mitochondria. Recent studies indicate that autophagy activation is defective in aging and osteoarthritis (OA), contributing to the cell death and tissue damage. In addition, there is increasing evidence that mitochondrial dysfunction plays an important role in OA pathogenesis. The objective of this study is to determine whether activation of autophagy protects from mitochondrial dysfunction in human chondrocytes. Methods Human chondrocytes were treated with Oligomycin, an inhibitor of mitochondrial respiratory chain (MRC) complex V. Autophagy activation was analyzed by determination of LC3-II, a marker for autophagosome formation. To investigate whether autophagy protects from mitochondrial dysfunction, autophagy was induced by mammalian target of rapamycin complex 1 (mTORC1) selective inhibitor Rapamycin and the dual mTORC1 and mTORC2 inhibitor Torin 1. SiAtg5 was employed to evaluate the role of autophagy in mitochondrial dysfunction. Results Mitochondrial dysfunction was induced by treatment with Oligomycin, which significantly decreased mitochondrial membrane potential (Δψm). This was associated with increased ROS production and cell death. Autophagy activation, reflected by LC3-II, was decreased in a time dependent manner. To evaluate whether autophagy regulates mitochondrial function, chondrocytes were pre-treated with Rapamycin and Torin 1 before Oligomycin. Autophagy activation significantly protected against mitochondrial dysfunction. Conversely, genetic inhibition of autophagy induced significant mitochondrial function defects. Conclusion Our data highlight the role of autophagy as a critical protective mechanism against mitochondrial dysfunction. Pharmacological interventions that enhance autophagy may have chondroprotective activity in cartilage degenerative processes such as OA.

These results suggest that decreased autophagy might be a mechanism by which diabetes influences cartilage degradation. Pharmacological activation of autophagy may be an effective therapeutic approach to prevent T2D-induced cartilage damage.

Our findings indicate that diabetic mice exhibit increased joint damage after experimental OA, and that autophagy activation might be an effective therapy for diabetes-accelerated OA.

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Phosphorylation of proteins pertinent to IGF-1 and MAPK signaling pathways were analyzed by immunoblotting. Hyperoxidized PRXs resulting from excessive ROS were detected using an antibody that reacts with any PRX family member when it is in the PRX-SO2/3 state. Reduced and disulfide oxidized PRX2 (cytosolic) and PRX3 (mitochondrial) were measured by separating the reduced monomers and oxidized dimers using non-reducing SDS-PAGE followed by immunoblotting with isoform-specific antibodies as described by Cox et al, 2010. Results: In time course studies, cell death after menadione treatment was first seen at 3 hrs (38% cell death) with 72% of the cells dead by 9 hrs. Cell death was completely inhibited when MCAT was overexpressed, thus confirming that the stimuli inducing cell death was mitochondrial in origin. Chondrocytes from donors of different ages were treated with menadione for 0-60 minutes in order to examine redox events leading to cell death. Hyperoxidized PRXs were observed with greater amounts seen in cells from older donors (Fig. 1). We further examined specific isoforms of PRX and determined if chondrocytes from older adults were more susceptible to induction of oxidative stress. A significant difference in PRX oxidation was noted between older (avg 63yrs) and younger (avg 38yrs) donors for PRX2 and PRX3 (Fig. 1). IGF-1 significantly increased phosphorylation of Akt at 5 minutes which was maintained out to 90 minutes but was significantly inhibited in cells pre-treated for 30 minutes with menadione (Fig. 2). Conversely, menadione treatment induced a sustained and significant increase in phosphorylation of the MAP kinases p38 (Fig. 2) and ERK. MCAT blocked menadione induced inhibition of Akt phosphorylation and reduced phosphorylation of catabolic p38.

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BackgroundOsteoarthritis (OA) is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation. Autophagy is essential to maintain chondrocyte homeostasis by regulating the intracellular macromolecule and organelle turnover (1). Previous findings indicated that autophagy is defective in Aging and OA articular cartilage (1,2), but the specific target/-s that regulates this mechanism and affect cartilage integrity are still unknown.ObjectivesThe objective of study is to identify relevant targets regulating autophagy in Aging and OA by proteomics.MethodsPrimary human chondrocytes from human donors were transfected with siRNA for Atg5 (100 nM, 72 hours), a key autophagy marker, to block the autophagy pathway. To identify the key proteins responding to defective autophagy, we performed a quantitative proteomics analysis of autophagy-deficient human chondrocytes using labeling iTRAQ (isobaric tags for relative and absolute quantitation) coupled with on-line 2D LC/MS/MS. Protein identification and quantification were performed using Protein Pilot Software v4.0 (ABSciex). Each MS/MS spectrum was searched in the Uniprot/Swissprot database for Homo sapiens. To confirm the candidate targets identified by the proteomic screening, inmortalized human chondrocytes (Tc28a2), human cartilage from healthy, aged and osteoarthritis human patients and mouse knee joints from young and old mice were employed to perform Western Blot and Histology analysis, respectively. The candidate targets were: Atg5 and LC3 for constitutive autophagy, p62, as a defective autophagy marker, Lamin A/C as an aging marker.ResultsFrom the total of 599 proteins found, 21 were significantly altered (p<0.05) in at least two donors from a total number of three. However, Lamin A/C, a nuclear protein implicated in premature cell senescence, was significantly upregulated in all the donors (p<0.05). To validate these results, TC28a2 human chondrocytes were transfected with siAtg5. Then, the expression of Atg5, LC3, p62, and Lamin A/C was evaluated. The results indicated a reduction in autophagy expression, accompanied with an increase in aging marker expression. Importantly, in human cartilage from both aged and OA patients, autophagy markers were significantly downregulated and Lamin A/C expression was upregulated, compared to healthy cartilage. Furthermore, articular cartilage from young mice (4 months old) and old mice (28 months old) was studied, suggesting that autophagy loss-of-function is correlated with premature senescence in articular cartilage.ConclusionsProteomics analysis of joint cells and tissue has revealed features of premature senescence when autophagy is disrupted in chondrocytes and cartilage. Lamin A/C, was identified as candidate target for regulating cartilage function in situations of defective autophagy, including aging and OA. These results support the hypothesis that autophagy is decreased with aging, and represents a key mechanism in the development of cartilage degradation.ReferencesCaramés B., et al.,...

BackgroundAutophagy, a key cellular quality control mechanism, is defective in Osteoarthritis (OA) and Type 2 Diabetes (T2D) (1,2). T2D has been proposed as a risk factor for OA. Although epidemiological studies suggest a strong association between these diseases (3), how T2D may have an effect on the deterioration of articular cartilage is still unknown.ObjectivesThe objective of this study is to understand the role of autophagy in the articular cartilage function under diabetic conditions.MethodsHuman chondrocyte cell line (TC28a2) and primary human chondrocytes (HC) were cultivated in DMEM high glucose (25 mM) and treated with Insulin (10, 100, 500 nM) for 2, 6 and 24 hours. Activity of LC3-II, Akt and rpS6 was evaluated by Western blotting (WB). To investigate whether autophagy activation protects from diabetic conditions, autophagy was induce by Rapamycin (10 μM). Human cartilage explants were cultivated in DMEM 25mM glucose and insulin (100, 500, 1000nM) for 24 hours to evaluate histopathological changes. MMP-13 and IL-1β expression was determined by immunohistochemistry and WB, respectively. Expression of LC3 and p-rpS6 was determined by WB in human chondrocytes from Non Diabetic-OA and Diabetic-OA patients.ResultsIn the presence of high glucose and increased doses of insulin autophagy was decreased in a dose dependent-manner in human chondrocytes, as indicated by LC3II expression, the main marker of autophagy activation (TC28-a2; p<0.05 at 6 hours post-treatment; HC; p<0.01 at 24 hours post-treatment). To investigate the mechanism by which autophagy is reduced by insulin, Akt and rpS6 phosphorylation was analyzed. We observed a significant increase in p-AKT and p-rpS6 activity, suggesting that insulin effect is mediated by AKT/mTOR pathway (TC28-a2 p<0.05 at 6 hours; HC; p<0.01 at 2 hours). Autophagy activation by Rapamycin reversed insulin effects on LC3 and p-rbS6 expression (Tc28a2 and HC:p<0.05), indicating that autophagy induction prevents insulin-mediated autophagy signaling downregulation. To evaluate the impact of insulin-mediated autophagy regulation in the context of articular cartilage biology, cartilage explants were treated with insulin (100, 500 and 1000 nM) for 24 hours. Histological analysis indicated a loss of proteoglycans and increased MMP-13 and IL-1β expression (p<0.01) after insulin treatment. Remarkably, chondrocytes from OA-diabetic patients showed decreased LC3 and increased p-rpS6 expression compared to Non-Diabetic OA patients.ConclusionsOur findings demonstrate that diabetic conditions decrease autophagy by an AKT/mTOR dependent mechanism. Pharmacological activation of autophagy might protect against T2D in human chondrocytes. Our data also indicate that chondrocytes from OA-diabetic patients exhibit a deficient autophagy. Taking together, these results suggest that impaired autophagy might be one of the mechanisms by which T2D diabetes accelerates cartilage degradation.ReferencesCaramés, B., et al., Arthritis Rheum, 2010. 62(3): p. 791-801Murrow, L., et al. Annu Rev Pathol, 201...