Purpose: An underlying cause of osteoarthritis (OA) is the inability of chondrocytes to maintain homeostasis in response to changing stress conditions. The purpose of this article was to review and experimentally evaluate oxidative stress resistance and resilience concepts in cartilage using glutathione redox homeostasis as an example. This framework may help identify novel approaches for promoting chondrocyte homeostasis during aging and obesity. Materials and Methods:Changes in glutathione content and redox ratio were evaluated in three models of chondrocyte stress: 1) age-and tissue-specific changes in joint tissues of 10 and 30-month old F344BN rats, including ex vivo patella culture experiments to evaluate Nacetylcysteine dependent resistance to an interleukin-1beta stress; 2) effect of different bout durations and patterns of cyclic compressive loading in bovine cartilage on glutathione stress resistance and resilience pathways; 3) time-dependent changes in GSH:GSSG in primary chondrocytes from wild-type and Sirt3 deficient mice challenged with the pro-oxidant menadione.Results: Glutathione was more abundant in cartilage than meniscus or infrapatellar fat pad, although cartilage was also more susceptible to age-related glutathione oxidation. Glutathione redox homeostasis was sensitive to the duration of compressive loading such that load-induced oxidation required unloaded periods to recover and increase total antioxidant capacity. Exposure to a pro-oxidant stress enhanced stress resistance by increasing glutathione content and GSH:GSSG ratio, especially in Sirt3 deficient cells. However, the rate of recovery, a marker of resilience, was delayed without Sirt3.Conclusions: OA-related models of cartilage stress reveal multiple mechanisms by which glutathione provides oxidative stress resistance and resilience.

The Ran GTPase regulates nuclear import and export by controlling the assembly state of transport complexes. This involves the direct action of RanGTP, which is generated in the nucleus by the chromatin‐associated nucleotide exchange factor, RCC1. Ran interactions with RCC1 contribute to formation of a nuclear:cytoplasmic (N:C) Ran protein gradient in interphase cells. In previous work, we showed that the Ran protein gradient is disrupted in fibroblasts from Hutchinson–Gilford progeria syndrome (HGPS) patients. The Ran gradient disruption in these cells is caused by nuclear membrane association of a mutant form of Lamin A, which induces a global reduction in heterochromatin marked with Histone H3K9me3 and Histone H3K27me3. Here, we have tested the hypothesis that heterochromatin controls the Ran gradient. Chemical inhibition and depletion of the histone methyltransferases (HMTs) G9a and GLP in normal human fibroblasts reduced heterochromatin levels and caused disruption of the Ran gradient, comparable to that observed previously in HGPS fibroblasts. HMT inhibition caused a defect in nuclear localization of TPR, a high molecular weight protein that, owing to its large size, displays a Ran‐dependent import defect in HGPS. We reasoned that pathways dependent on nuclear import of large proteins might be compromised in HGPS. We found that nuclear import of ATM requires the Ran gradient, and disruption of the Ran gradient in HGPS causes a defect in generating nuclear γ‐H2AX in response to ionizing radiation. Our data suggest a lamina–chromatin–Ran axis is important for nuclear transport regulation and contributes to the DNA damage response.

Understanding how obesity‐induced metabolic stress contributes to synovial joint tissue damage is difficult because of the complex role of metabolism in joint development, maintenance, and repair. Chondrocyte mitochondrial dysfunction is implicated in osteoarthritis (OA) pathology, which motivated us to study the mitochondrial deacetylase enzyme sirtuin 3 (Sirt3). We hypothesized that combining high‐fat‐diet (HFD)‐induced obesity and cartilage Sirt3 loss at a young age would impair chondrocyte mitochondrial function, leading to cellular stress and accelerated OA. Instead, we unexpectedly found that depleting cartilage Sirt3 at 5 weeks of age using Sirt3‐flox and Acan‐CreERT2 mice protected against the development of cartilage degeneration and synovial hyperplasia following 20 weeks of HFD. This protection was associated with increased cartilage glycolysis proteins and reduced mitochondrial fatty acid metabolism proteins. Seahorse‐based assays supported a mitochondrial‐to‐glycolytic shift in chondrocyte metabolism with Sirt3 deletion. Additional studies with primary murine juvenile chondrocytes under hypoxic and inflammatory conditions showed an increased expression of hypoxia‐inducible factor (HIF‐1) target genes with Sirt3 deletion. However, Sirt3 deletion impaired chondrogenesis using a murine bone marrow stem/stromal cell pellet model, suggesting a context‐dependent role of Sirt3 in cartilage homeostasis. Overall, our data indicate that Sirt3 coordinates HFD‐induced changes in mature chondrocyte metabolism that promote OA. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Objective Obesity accelerates the development of osteoarthritis (OA) during aging and is associated with altered chondrocyte cellular metabolism. Protein lysine malonylation (MaK) is a posttranslational modification (PTM) that has been shown to play an important role during aging and obesity. The objective of this study was to investigate the role of sirtuin 5 (Sirt5) in regulating MaK and cellular metabolism in chondrocytes under obesity-related conditions. Methods MaK and SIRT5 were immunostained in knee articular cartilage of obese db/db mice and different aged C57BL6 mice with or without destabilization of the medial meniscus surgery to induce OA. Primary chondrocytes were isolated from 7-day-old WT and Sirt5−/− mice and treated with varying concentrations of glucose and insulin to mimic obesity. Sirt5-dependent effects on MaK and metabolism were evaluated by western blot, Seahorse Respirometry, and gas/chromatography-mass/spectrometry (GC-MS) metabolic profiling. Results MaK was significantly increased in cartilage of db/db mice and in chondrocytes treated with high concentrations of glucose and insulin (GluhiInshi). Sirt5 was increased in an age-dependent manner following joint injury, and Sirt5 deficient primary chondrocytes had increased MaK, decreased glycolysis rate, and reduced basal mitochondrial respiration. GC-MS identified 41 metabolites. Sirt5 deficiency altered 13 distinct metabolites under basal conditions and 18 metabolites under GluhiInshi treatment. Pathway analysis identified a wide range of Sirt5-dependent altered metabolic pathways that include amino acid metabolism, TCA cycle, and glycolysis. Conclusion This study provides the first evidence that Sirt5 broadly regulates chondrocyte metabolism. We observed changes in SIRT5 and MaK levels in cartilage with obesity and joint injury, suggesting that the Sirt5-MaK pathway may contribute to altered chondrocyte metabolism that occurs during OA development.

ObjectiveObesity accelerates the development of osteoarthritis (OA) during aging and is associated with altered chondrocyte cellular metabolism. The objective of this study was to investigate the role of sirtuin 5 (SIRT5) in regulating chondrocyte protein lysine malonylation (MaK) and cellular metabolism under obesity-related conditions.MethodsMaK and SIRT5 were immunostained in knee articular cartilage of obese db/db mice and different aged C57BL6 mice with or without destabilization of the medial meniscus (DMM) surgery to induce OA. Primary chondrocytes were isolated from 7-day-old WT and Sirt5−/− mice and treated with varying concentrations of glucose and insulin to mimic obesity. Sirt5-dependent effects on MaK and metabolism were evaluated by Western blot, Seahorse Respirometry, and gas/chromatography-mass/spectrometry (GC-MS) metabolic profiling.ResultsMaK was significantly increased in cartilage of db/db mice and in chondrocytes treated with high concentrations of glucose and insulin (GluhiInshi). Sirt5 protein was increased in an age-dependent manner following joint injury, and Sirt5 deficient primary chondrocytes had increased MaK, decreased glycolysis rate, and reduced basal mitochondrial respiration. GC-MS identified 41 metabolites. Sirt5 deficiency altered 13 distinct metabolites under basal conditions and 18 metabolites under GluhiInshi treatment. Pathway analysis identified a wide range of Sirt5-dependent altered metabolic pathways that include amino acid metabolism, TCA cycle, and glycolysis.ConclusionThis study provides the first evidence that Sirt5 broadly regulates chondrocyte metabolism. We observed changes in Sirt5 and MaK levels in cartilage with obesity and joint injury, suggesting that the Sirt5-MaK pathway may contribute to altered chondrocyte metabolism that occurs during OA development.

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Purpose: Cellular senescence is a phenotypic state that emerges in response to stress after injury and during aging. Senescent cells are characterized by increased expression of the cell cycle inhibitor p16 INK4a (p16), high lysosomal activity, and the production of pro-inflammatory and matrix-degrading proteins known as the senescence associated secretory phenotype (SASP). Expression of p16 increases in both murine and human chondrocytes with age and high p16 expression correlates with SASP factor production. Furthermore, treatment with "senolytic" compounds that specifically target senescent cells for apoptosis was shown to mitigate post-traumatic OA in a murine model. The aim of this study was to establish a cartilage explant model system to explore the functional effects of selectively eliminating p16-expressing (senescent) chondrocytes. Methods: A knock-in murine genetics approach was used to replace the coding region of p16 with the fluorescent molecule tdTomato in order to quantify p16 expression on a single-cell basis with flow cytometry. Femoral cap cartilage explants were isolated from mice with one intact copy of p16 and one copy of the p16 tdTomato allele. Explants were cultured for three weeks either in control media or senescence induction media containing 1 ng/ml transforming growth factor beta-1 and 5 ng/ ml basic fibroblastic growth factor. Explants were digested and the percentage of p16 tdTomato -high cells was determined by flow cytometry. Cell sorting was used to separate chondrocytes into p16 tdTomato -high and p16 tdTomato -low fractions and these populations were analyzed for senescence features such as senescence-associated beta galactosidase (SABG) activity. Senescent cells upregulate anti-apoptotic pathways to maintain survival despite significant cellular damage. The ability to selectively eliminate p16-high cells with a drug that inhibits this apoptotic block is both a potential treatment and a way to further confirm the senescent nature of the p16 tdTomato -high cell fraction. In this study, senolytic treatment was performed using navitoclax, a BCL pathway inhibitor, at a concentration of 5 mM for 72 hours. As an initial assessment of how senolytic clearance affects cartilage health, the glycosaminoglycans (GAGs) content was measured in femoral cap explants that were cultured for an additional week following control or navitoclax treatment. Results: The treatment of murine cartilage explants with growth factors for three weeks induces a significant fraction of p16 tdTomato -high chondrocytes (Figure 1). This p16 tdTomato -high cell population demonstrates higher SABG activity as compared to the p16 tdTomato -low fraction (n¼4 sorts, mean ¼ 39.1% vs. 20.5% positive, p¼0.03 by paired t-test). Following treatment with the senolytic navitoclax, explants showed a significant reduction in the percentage of p16 tdTomato -high cells (Figure 2). As compared to control treatment, clearing senescent cells with navitoclax shows a trend towards increasing the GAG content per cell in cartilage exp...