ObjectivesCircadian rhythm (CR) was identified by RNA sequencing as the most dysregulated pathway in human osteoarthritis (OA) in articular cartilage. This study examined circadian rhythmicity in cultured chondrocytes and the role of the CR genes NR1D1 and BMAL1 in regulating chondrocyte functions.MethodsRNA was extracted from normal and OA-affected human knee cartilage (n = 14 each). Expression levels of NR1D1 and BMAL1 mRNA and protein were assessed by quantitative PCR and immunohistochemistry. Human chondrocytes were synchronized and harvested at regular intervals to examine circadian rhythmicity in RNA and protein expression. Chondrocytes were treated with small interfering RNA (siRNA) for NR1D1 or BMAL1, followed by RNA sequencing and analysis of the effects on the transforming growth factor beta (TGF-β) pathway.ResultsNR1D1 and BMAL1 mRNA and protein levels were significantly reduced in OA compared to normal cartilage. In cultured human chondrocytes, a clear circadian rhythmicity was observed for NR1D1 and BMAL1. Increased BMAL1 expression was observed after knocking down NR1D1, and decreased NR1D1 levels were observed after knocking down BMAL1. Sequencing of RNA from chondrocytes treated with NR1D1 or BMAL1 siRNA identified 330 and 68 significantly different genes, respectively, and this predominantly affected the TGF-β signaling pathway.ConclusionsThe CR pathway is dysregulated in OA cartilage. Interference with circadian rhythmicity in cultured chondrocytes affects TGF-β signaling, which is a central pathway in cartilage homeostasis.

ObjectiveAutophagy is a cellular homeostasis mechanism that facilitates normal cell function and survival. Objectives of this study were to determine associations between autophagic responses with meniscus injury, joint aging, and osteoarthritis (OA), and to establish the temporal relationship with structural changes in menisci and cartilage.MethodsConstitutive activation of autophagy during aging was measured in GFP-LC3 transgenic reporter mice between 6 and 30 months. Meniscus injury was created by surgically destabilizing the medial meniscus (DMM) to induce posttraumatic OA in C57BL/6J mice. Levels of autophagy proteins and activation were analyzed by confocal microscopy and immunohistochemistry. Associated histopathological changes, such as cellularity, matrix staining, and structural damage, were graded in the meniscus and compared to changes in articular cartilage.ResultsIn C57BL/6J mice, basal autophagy was lower in the meniscus than in articular cartilage. With increasing age, expression of the autophagy proteins ATG5 and LC3 was significantly reduced by 24 months. Age-related changes included abnormal Safranin-O staining and reduced cellularity, which preceded structural damage in the meniscus and articular cartilage. In mice with DMM, autophagy was induced in the meniscus while it was suppressed in cartilage. Articular cartilage exhibited the most profound changes in autophagy and structure that preceded meniscus degeneration. Systemic administration of rapamycin to mice with DMM induced autophagy activation in cartilage and reduced degenerative changes in both meniscus and cartilage.ConclusionAutophagy is significantly affected in the meniscus during aging and injury and precedes structural damage. Maintenance of autophagic activity appears critical for meniscus and cartilage integrity.

ObjectivesAging is an important osteoarthritis (OA) risk factor and compromised stress defense responses may mediate this risk. The Sestrins (Sesn) promote cell survival under stress conditions and regulate AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signaling. This study examined Sesn expression in normal and OA cartilage and functions of Sesn in chondrocytes.MethodsSesn expression in human and mouse normal and OA cartilage was analyzed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. Sesn function was investigated by using small interfering RNA (siRNA) mediated Sesn knockdown and overexpression with analysis of cell survival, gene expression, autophagy, and AMPK and mTOR activation.ResultsSesn mRNA levels were significantly reduced in human OA cartilage and immunohistochemistry of human and mouse OA cartilage also showed a corresponding reduction in protein levels. In cultured human chondrocytes Sesn1, 2 and 3 were expressed and increased by tunicamycin, an endoplasmic reticulum (ER) stress response inducer and 2-deoxyglucose (2DG), a metabolic stress inducer. Sesn1 and 2 were increased by tBHP, an oxidative stress inducer.Sesn knockdown by siRNA reduced chondrocyte viability under basal culture conditions and in the presence of 2DG. Sesn overexpression enhanced LC3-II formation and autophagic flux, and this was related to changes in mTOR but not AMPK activation.ConclusionThese findings are the first to show that Sesn expression is suppressed in OA affected cartilage. Sesn support chondrocyte survival under stress conditions and promote autophagy activation through modulating mTOR activity. Suppression of Sesn in OA cartilage contributes to deficiency in an important cellular homeostasis mechanism.

Osteoarthritis (OA) is the most prevalent joint disease, but neither preventive measures nor disease-modifying drugs are available and a continuing need exists for safe and effective symptom-modifying therapies. Clinical trials of candidate disease-modifying OA drugs in patients with established or advanced disease have not demonstrated their efficacy, but these failed trials have motivated investigation into the mechanisms that maintain joint health. The enhancement of such mechanisms could be a novel approach to reducing the risk of OA. Aging is one of the most important risk factors for OA; however, aging of joint cartilage is a process that is distinct from the subsequent cartilage changes that develop following the onset of OA. This Review focuses on the mechanisms that maintain cell and tissue homeostasis, and how these mechanisms fail during the aging process. Autophagy is a cellular homeostasis mechanism for the removal of dysfunctional organelles and macromolecules. Defective autophagy is involved in the pathogenesis of aging-related diseases and recent observations indicate that this process is compromised in aging cartilage. Augmentation of homeostasis mechanisms is discussed as a novel avenue to delay joint aging and reduce OA risk.

The balance between anabolic and catabolic signaling pathways is critical in maintaining cartilage homeostasis and its disturbance contributes to joint diseases such as osteoarthritis (OA). A unique mechanism that modulates the activity of cell signaling pathways is controlled by extracellular heparan endosulfatases Sulf-1 and Sulf-2 (Sulfs) that are overexpressed in OA cartilage. This study addressed the role of Sulfs in cartilage homeostasis and in regulating bone morphogenetic protein (BMP)/Smad and fibroblast growth factor (FGF)/Erk signaling in articular cartilage. Spontaneous cartilage degeneration and surgically induced OA were significantly more severe in Sulf-1^−/− and Sulf-2^−/− mice compared with wild-type mice. MMP-13, ADAMTS-5, and the BMP antagonist noggin were elevated whereas col2a1 and aggrecan were reduced in cartilage and chondrocytes from Sulf^−/− mice. Articular cartilage and cultured chondrocytes from Sulf^−/− mice showed reduced Smad1 protein expression and Smad1/5 phosphorylation, whereas Erk1/2 phosphorylation was increased. In human chondrocytes, Sulfs siRNA reduced Smad phosphorylation but enhanced FGF-2-induced Erk1/2 signaling. These findings suggest that Sulfs simultaneously enhance BMP but inhibit FGF signaling in chondrocytes and maintain cartilage homeostasis. Approaches to correct abnormal Sulf expression have the potential to protect against cartilage degradation and promote cartilage repair in OA.

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.

The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis. Mechanisms that regulate the unique phenotype of SZ chondrocytes and maintain SZ integrity are unknown. We recently demonstrated that expression of the chromatin protein high mobility group box (HMGB) protein 2 is restricted to the SZ in articular cartilage suggesting a transcriptional regulation involving HMGB2 in SZ. Here, we show that an interaction between HMGB2 and the Wnt/β-catenin pathway regulates the maintenance of the SZ. We found that the Wnt/β-catenin pathway is active specifically in the SZ in normal mouse knee joints and colocalizes with HMGB2. Both Wnt signaling and HMGB2 expression decrease with aging in mouse joints. Our molecular studies show that HMGB2 enhances the binding of Lef-1 to its target sequence and potentiates transcriptional activation of the Lef-1-β-catenin complex. The HMG domain within HMGB2 is crucial for interaction with Lef-1, suggesting that both HMGB2 and HMGB1 may be involved in this function. Furthermore, conditional deletion of β-catenin in cultured mouse chondrocytes induced apoptosis. These findings define a pathway where protein interactions of HMGB2 and Lef-1 enhance Wnt signaling and promote SZ chondrocyte survival. Loss of the HMGB2-Wnt signaling interaction is a new mechanism in aging-related cartilage pathology.

ObjectiveAging is a main risk factor for the development of osteoarthritis (OA) and the molecular mechanisms underlying the aging-related changes in articular cartilage include increased mammalian target of rapamycin (mTOR) signaling and defective autophagy. REDD1 is an endogenous inhibitor of mTOR that regulates cellular stress responses. In this study we measured REDD1 expression in normal, aged and OA cartilage and assessed REDD1 function in human and mouse articular chondrocytes.MethodsREDD1 expression was analyzed in human and mouse articular cartilage by qPCR, western blotting, and immunohistochemistry. For functional studies, REDD1 and TXNIP knockdown or overexpression was performed in chondrocytes in the presence or absence of rapamycin and chloroquine, and mTOR signaling and autophagy were measured by western blotting. REDD1/TXNIP protein interaction was assessed by co-immunoprecipitation experiments.ResultsHuman and mouse cartilage from normal knee joints expressed high levels of REDD1. REDD1 expression was significantly reduced in aged and OA cartilage. In cultured chondrocytes, REDD1 knockdown increased whereas REDD1 overexpression decreased mTOR signaling. In addition, REDD1 activated autophagy by an mTOR independent mechanism that involved protein/protein interaction with TXNIP. The REDD1/TXNIP complex was required for autophagy activation in chondrocytes.ConclusionThe present study shows that REDD1 is highly expressed in normal human articular cartilage and reduced during aging and OA. REDD1 in human chondrocytes negatively regulates mTOR activity and is essential for autophagy activation. Reduced REDD1 expression thus represents a novel mechanism for the increased mTOR activation and defective autophagy observed in OA.

ObjectiveAging is a major risk factor for osteoarthritis (OA). Forkhead-box class O (FoxO) transcription factors regulate mechanisms of cellular aging, including protein quality control, autophagy and defenses against oxidative stress. The objective of this study was to analyze FoxO transcription factors in normal, aging and OA cartilage.DesignKnee joints from humans ages 23–90 and from mice at the age of 4–24 months and following surgically induced OA were analyzed for expression of FoxO proteins. Regulation of FoxO protein expression and activation was analyzed in cultured chondrocytes.ResultsHuman cartilage expressed FOXO1 and FOXO3 but not FOXO4 proteins. FOXO1 and FOXO3 were more strongly expressed the superficial and mid zone as compared to the deep zone and were mainly localized in nuclei. During human joint aging, expression of FOXO1 and FOXO3 was markedly reduced in the superficial zone of cartilage regions exposed to maximal weight bearing. In OA cartilage, chondrocyte clusters showed strong FOXO phosphorylation and cytoplasmic localization. Similar patterns of FOXO expression in normal joints and changes in aging and OA were observed in mouse models. In cultured chondrocytes, IL-1β and TNF-α suppressed FOXO1, while TGF-β and PDGF increased FOXO1 and FOXO3 expression. FOXO1 and FOXO3 phosphorylation was increased by IL-1β, PDGF, bFGF, IGF-1, and the oxidant t-BHP.ConclusionsNormal articular cartilage has a tissue specific signature of FoxO expression and activation and this is profoundly altered in aging and OA in humans and mice. Changes in FoxO expression and activation may be involved in cartilage aging and OA.

This review is focused on aging-related changes in cells and extracellular matrix of the articular cartilage. Major extracellular matrix changes are a reduced thickness of cartilage, proteolysis, advanced glycation and calcification. The cellular changes include reduced cell density, cellular senescence with abnormal secretory profiles, and impaired cellular defense mechanisms and anabolic responses. The extracellular and cellular changes compound each other, leading to biomechanical dysfunction and tissue destruction. The consequences of aging-related changes for joint homeostasis and risk for osteoarthritis are discussed.This article is part of a Special Issue entitled “Osteoarthritis”.

ObjectiveTo establish a standardized protocol for histopathological assessment of murine menisci that can be applied to evaluate transgenic, knock-out/in, and surgically induced OA models.MethodsKnee joints from C57BL/6J mice (6–36 months) as well as from mice with surgically-induced OA were processed and cut into sagittal sections. All sections included the anterior and posterior horns of the menisci and were graded for (1) surface integrity, (2) cellularity, (3) Safranin-O staining distribution and intensity. Articular cartilage in the knee joints was also scored.ResultsThe new histopathological grading system showed good inter- and intra-class correlation coefficients. The major age-related changes in murine menisci in the absence of OA included decreased Safranin O staining intensity, abnormal cell distribution and the appearance of acellular areas. Menisci from mice with surgically-induced OA showed severe fibrillations, partial/total loss of tissue, and calcifications. Abnormal cell arrangements included both regional hypercellularity and hypocellularity along with hypertrophy and cell clusters. In general, the posterior horns were less affected by age and OA.ConclusionA new standardized protocol and histopathological grading system has been developed and validated to allow for a comprehensive, systematic evaluation of changes in aging and OA-affected murine menisci. This system was developed to serve as a standardized technique and tool for further studies in murine meniscal pathophysiology models.