Disruption of the articular cartilage surface, degradation of extracellular matrix (ECM), and reduced cartilage cellularity are major histologic features of osteoarthritis (OA), the most common aging-related joint pathology (1, 2). Because chondrocytes maintain the dynamic equilibrium between production of the ECM and its enzymatic degradation, it is important to uncover the molecular mechanisms that control cell fate in cartilage (3). Many factors can be implicated in the development of OA, but one of the most important risk factors is aging (4). Aging is a process characterized by a progressive accumulation of damaged macromolecules and organelles in somatic cells during the postdevelopmental period, leading to the decreased ability of cells to function normally and survive (5). However, mechanisms leading to aging-related cartilage degeneration remain to be determined.
Macroautophagy is a major physiologic mechanism that targets altered and dysfunctional cytosolic macromolecules, membranes, and organelles for delivery to lysosomes for degradation and recycling (5–8). Atg genes control the autophagy process leading to the induction and nucleation of autophagic vesicles as well as to their expansion and fusion with lysosomes, allowing enzymatic degradation and recycling (9, 10). Among the Atg genes, Atg1, Atg6, and Atg8 (Unc-51–like kinase 1 [ULK1], Beclin1, and microtubule-associated protein 1 light chain 3 [LC3], respectively, in mammals) are 3 major regulators of the autophagy pathway. ULK1 is a key intermediate in the transduction of proautophagic signals to autophagosome formation (11). Beclin1 forms a complex with type III phosphatidylinositol 3-kinase and Vps34 that allows nucleation of the autophagic vesicle (12). Finally, the formation and expansion of the autophagosome requires 2 protein conjugation systems that involve the Atg proteins LC3 and Atg12 (13).
LC3 is present in 2 forms, LC3-I in the cytoplasm and LC3-II bound to the autophagosome membrane. During autophagy, LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system, resulting in the association of LC3-II with autophagy vesicles. The amount of LC3-II is correlated with the extent of autophagosome formation (14). Autophagy plays a fundamental role in cellular homeostasis and functions primarily to promote cellular and organism health. In certain physiologic and pathologic conditions, it can also lead to a form of cell death that is characterized by cytoplasmic vacuolation and is termed type II programmed cell death or cell death by autophagy (15–17). In most experimental models, suppression of autophagy genes leads to cell death, indicating a protective and survival-promoting function of autophagy (18–20).
In articular cartilage, which is characterized by a very low rate of cell turnover (21), this mechanism would appear to be essential to maintain cellular integrity, function, and survival. Furthermore, while autophagy changes in various models and tissues with aging (22), this has not been investigated in articular cartilage. In the present study, we demonstrate that autophagy is a constitutively active and apparently protective process for maintenance of the homeostatic state 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 in articular cartilage. Furthermore, the reduction of these key regulators of autophagy is accompanied by an increase in apoptosis. These results suggest that compromised autophagy may contribute to the development of OA.
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The relationship between aging and OA is clinically and epidemiologically evident, and recent findings provide insight into mechanisms that lead to aging-related changes in cells and ECM (30). Articular cartilage appears to be highly susceptible to the accumulation of aging-related changes, in part due to the relatively low turnover of ECM and cells. It has been estimated that the half-life of type II collagen is ∼117 years (31), and various measurements in mature cartilage revealed only a very small fraction of proliferating cells (32). Thus, cartilage matrix and cells are prone to accumulating changes related to trauma or to mechanical or oxidant stress over time (33). At the cellular level, a large number of aging-related changes have been documented. These include an overall reduction in cartilage cellularity, reduced antioxidant defenses, altered responses to growth factors and cytokines, and changes in gene and protein expression patterns (21). Physiologic mechanisms that allow for repair of cellular damage are thus critical for the maintenance of chondrocyte survival and function. Best characterized in this regard are mechanisms for nuclear and mitochondrial DNA repair. In contrast, mechanisms capable of replacing damaged proteins and organelles in chondrocytes are essentially unknown.
Autophagy has gained interest in the past decade due to its role in the pathogenesis of various diseases and in regulation of the aging process. Autophagy has been characterized as a highly regulated cellular mechanism with both beneficial and pathogenic effects (34). Normal cellular development and growth require a well-regulated balance between protein synthesis and degradation. Eukaryotic cells have 2 major mechanisms for degradation, the proteasome and autophagy pathways. Autophagy is involved in the bulk degradation of long-lived cytosolic proteins and organelles, whereas the ubiquitin proteasome system degrades specific short-lived proteins (35). Recent studies provide compelling evidence that, at least in model organisms, autophagy protects against diverse pathologies, such as neurodegeneration, heart diseases, infections, cancer, and aging (8). Genetic studies in mice support the importance of autophagy in physiologic and pathologic events. In fact, loss of autophagy genes leads to neurodegeneration, cardiomyopathies, and abnormalities in skeletal development and is associated with the accumulation of cytoplasmic protein aggregates (18, 36–39).
The mechanisms by which autophagy is lost with aging are mainly related to the failure of the lysosomal hydrolases, resulting in an increase of toxic protein products and slow clearance of autophagosomes in the aging tissues (40). In addition, other reports described alterations in the response of macroautophagy to hormonal changes with aging. In particular, the effects of oxidative stress on the insulin receptor signaling pathway seem to play a critical role in decreased autophagy in aged organisms (41). The signaling network involving longevity factors sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1), mechanistic target of rapamycin (mTOR), FoxO3, NF-κB, and p53 regulate autophagy and might have a role in the aging process. NF-κB and mTOR are repressors of the autophagy pathway under input signals of stress and inflammation, while SIRT1, a stress resistance and longevity factor, and FoxO3, a major regulator of cellular metabolism, proliferation, and stress resistance, enhance autophagy (42).
The present study demonstrates that ULK1, Beclin1, and LC3 are expressed in normal human and murine articular cartilage, suggesting activation of autophagy, and the presence of LC3-II directly indicates autophagosome formation (14). However, in OA cartilage and chondrocytes, the expression of these autophagy markers was significantly decreased. Importantly, the reduction in LC3-II implies defective or reduced autophagy in OA. These observations are consistent with the notion that basal autophagic activity decreases with age, thus contributing to the accumulation of damaged macromolecules and susceptibility to aging-related diseases (43). Abnormal protein aggregation and formation of characteristic pathologic structures are central features of such diseases. Interestingly, cellular responses to protein misfolding and aggregation are intimately related to mechanisms of pathogenesis and are potential targets of new therapies (44).
The present results also show that autophagy was decreased in the surgically induced OA model in mice. Since this was observed in relatively young mice (ages 2–4 months), it is apparently not a consequence of aging-related mechanisms. We found similar results in porcine cartilage explants following exposure to mechanical injury (Caramés B: unpublished observations). It will be of interest to determine mechanisms that are responsible for the suppression or loss of autophagy in the surgical OA model and after mechanical injury. However, both the surgical OA model and mechanically injured cartilage feature increased cell death, suggesting that loss of autophagy may contribute to cell death.
Little is known about the role of autophagy in articular cartilage. Morphologic changes similar to autophagic cell death have been described (45). More information is available in autophagy studies of the epiphyseal growth plate. Autophagy regulates maturation and promotes survival of terminally differentiated chondrocytes under conditions of stress and hypoxia (46–48). In fact, silencing the transcription factor hypoxia-inducible factor 1 (HIF-1) in chondrocytes decreases the expression of Beclin1, suggesting that this factor may have a role as a positive autophagy regulator and that it could play a protective role against cell death (48). In contrast, suppression of HIF-2 in OA and aging cartilage was associated with increased HIF-1 expression and autophagy induction (49).
The protective role of autophagy in endochondral ossification was further supported by observations that its inactivation leads to severe skeletal abnormalities, due in part to cell death (39). In addition, autophagy is essential for early embryonic development, supported by observations that Beclin1 disruption causes early embryonic lethality, autophagy deficiency, and apoptosis (50). In order to investigate the relationship between autophagy and apoptotic cell death as well as the mechanism of chondrocyte loss and cartilage degradation in aging and OA, immunohistochemistry was performed in the present study for the apoptosis marker PARP p85. We found that autophagy was decreased in the aging-related and surgically induced OA mouse models, while apoptotic cell death was increased. This correlation requires further analysis to determine whether a direct causal relationship exists between the 2 processes in cartilage.
In summary, this study is the first to demonstrate that autophagy may be a protective or homeostatic mechanism in normal cartilage. In contrast, human OA and spontaneous 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 may represent a novel mechanism in the development of OA.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Lotz had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Caramés, Blanco, Lotz.
Acquisition of data. Caramés, Taniguchi, Otsuki.
Analysis and interpretation of data. Caramés, Lotz.