Articular cartilage stability depends on the biosynthetic activities of chondrocytes, which counteract normal degradation of matrix macromolecules. Aging and the degeneration of articular cartilage in osteoarthritis (OA) are distinct processes; however, the incidence and prevalence of synovial joint degeneration increase dramatically in middle age (1) and the risk of posttraumatic OA following intraarticular fracture of the knee increases 3–4-fold after the age of 50 years (2), suggesting that age-related cartilage changes render the tissue incapable of adequately maintaining the extracellular matrix.
Previous investigations have indicated that replicative senescence of chondrocytes occurs in vivo (3–6). Studies of human articular chondrocytes from donors ranging in age from 1 year to 87 years showed that senescence-associated β-galactosidase (SA–β-gal) activity increased with advancing age, whereas both mitotic activity and mean telomere length (MTL) declined with age (5). This is indirect evidence in support of the hypothesis that age-related changes in articular cartilage increase vulnerability of the tissue to degeneration and that the association between OA and aging is due, at least in part, to features of chondrocyte senescence. Furthermore, increased SA–β-gal activity was observed in damaged OA cartilage, and MTL was shorter in cells near the lesion compared with distal sites in the same joint (6). These findings also suggest that features of chondrocyte senescence participate in the pathogenesis of OA. However, the exact mechanism of chondrocyte senescence and its implications with regard to OA pathogenesis remain unclear.
Cellular senescence is classified into 2 types: intrinsic senescence (telomere-dependent replicative senescence) and extrinsic premature senescence (telomere-independent senescence). It is thought that extrinsic senescence is induced by several types of stresses, such as oxidative stress, ultraviolet (UV) irradiation, or secretory factors (e.g., proinflammatory cytokines) (7–9). Also, mechanical and chemical stresses are well known to induce degeneration of articular cartilage. A variety of catabolic stresses involving mechanical loading, cytokines, and oxidative stress participate in the pathophysiology of OA. These catabolic factors may result in extrinsic stress–induced premature senescence of articular chondrocytes. If features of premature senescence in chondrocytes have an important role in the development and progression of OA, understanding of the mechanisms of chondrocyte down-regulation will be important for devising new approaches to the prevention and treatment of this disease.
Caveolae are vesicular invaginations of the plasma membrane. Caveolin 1 is the principal structural component of caveolae in vivo. Caveolin 2 has the same tissue distribution as caveolin 1 (10), while caveolin 3 is expressed only in striated muscle cell types (cardiac and skeletal) (11). It has been proposed that caveolins participate in vesicular trafficking events and signal transduction processes (12, 13), and several independent lines of evidence suggest that signaling molecules are sequestered, organized, and functionally regulated by caveolae microdomains (14, 15). Both caveolae and caveolin are expressed most abundantly in terminally differentiated cells, such as adipocytes, endothelial cells, and muscle cells. Interestingly, stress-induced cellular senescence is up-regulated by the expression of endogenous caveolin 1 (16, 17). These findings strongly indicate that caveolin 1 plays a central role in promoting stress-induced premature senescence. Recent work shows that caveolin 1 induces premature cellular senescence in response to various stress conditions, such as UV irradiation and oxidative stress, in primary cultures of fibroblasts, and that the senescent phenotype of fibroblasts can be reversed by reduction of caveolin 1 (17). These results suggest that caveolin 1 expression is closely involved in common stress-induced and age-related diseases.
Recently, caveolin 1 expression was demonstrated in normal human knee joint cartilage by immunohistochemical analysis (18). However, its role in joint cartilage remains unknown. Findings in the present study revealed, for the first time, that stress factors, i.e., interleukin-1β (IL-β) and oxygen free radicals, induce features of premature senescence of articular chondrocytes and that caveolin 1 mediates, at least in part, this process.
It has been reported that p38 MAPK is a senescence-executing molecule which is activated by both telomere-dependent and telomere-independent senescence-inducing stimuli (19), and that the ERK/MAPK pathway is responsible for Ras-induced senescence (20). The p53 tumor suppressor protein plays a critical role in regulating cell growth arrest (21). In addition, p21 mediates p53-dependent G1 arrest by inhibiting the activity of cyclin-dependent kinases (CDKs), which phosphorylate the retinoblastoma (Rb) gene product, as well as other substrates (22); unphosphorylated Rb does not release E2F and does not induce G1 entry into the cell cycle (23). We tried to elucidate the underlying signal transduction pathways involved in premature chondrocyte senescence induced by catabolic stresses. Features of chondrocyte senescence accelerated by catabolic stresses may contribute to the risk of cartilage degeneration by reducing chondrocyte viability.
- Top of page
- MATERIALS AND METHODS
Our results indicate that the catabolic stresses IL-1β and oxidative stress induce features of chondrocyte senescence through the overexpression of caveolin 1 in articular cartilage. Recently, attention has been focused on caveolin 1 overexpression–induced cellular senescence in human somatic cells (16, 17). Our findings of stress-induced expression of caveolin 1 and its involvement in features of chondrocyte senescence suggest that caveolin 1 has a role in the pathogenesis of articular cartilage degeneration in OA, by promoting stress-induced down-regulation of chondrocytes.
We observed expression of caveolin 1 in degenerated cartilage from rats with OA. Stronger expression of caveolin 1 in chondrocytes was found in central degenerated regions as compared with peripheral regions with less degeneration from the same articular cartilage samples. In samples (central degenerated region and peripheral region with less degeneration) from the same articular cartilage specimens from human OA patients, positivity for caveolin 1 was associated with histologic changes characteristic of OA. Furthermore, in rats with OA induced by transection of the ACL and MCL, our histologic analysis revealed an increase in caveolin 1 expression in chondrocytes, occurring prior to the progression of articular cartilage degeneration. Our findings in human articular cartilage tissue and in a rat OA model thus indicate for the first time that expression of caveolin 1 is involved in the progression of articular cartilage degeneration.
It has long been theorized that oxidative stress, resulting in the accumulation of proteins that have undergone oxidative damage, is the cause of aging-related changes in a number of tissues (29–31). Detection of nitrotyrosine, a marker of oxidative damage, in articular cartilage supports the notion of a role for oxidative damage to cartilage in the setting of both aging and OA (32). Articular chondrocytes actively produce endogenous reactive oxygen species, including nitric oxide (NO) (33), O2− (34), HO− (35), and H2O2 (36), suggesting the occurrence of oxidative stress in articular cartilage. In the present study, we confirmed that, although H2O2 in nontoxic levels (<100 μM) did not affect the viability of chondrocytes, chondrocytes showed the characteristic phenotypes of cellular senescence after exposure to oxidative stress (37).
This is the first reported study to demonstrate that features of chondrocyte senescence are induced by IL-1β. IL-1 has been shown both to inhibit chondrocyte anabolic activity, including proteoglycan synthesis (38), and to stimulate catabolic activity, including production of metalloproteinases (39). It has also been shown to stimulate chondrocyte expression of reactive oxygen species such as NO, which results in increased oxidative damage (40). Our finding of features of stress-induced senescence further extends knowledge of the roles of IL-1β in cartilage degeneration.
Cellular senescence represents an arrested state in which the cells remain viable but are not stimulated to divide by serum or passage in culture. In the present study, cell cycle analysis demonstrated that treatment with either IL-1β or H2O2 significantly induced chondrocyte arrest at the G0/G1 phase. This is further supported by evidence that IL-1β inhibits 3H-thymidine incorporation in cultured articular chondrocytes (41, 42). Our data suggest that H2O2 also increases the percentage of chondrocytes in the G2/M phase of the cell cycle; this phenomenon has also been found in fibroblasts after H2O2 stimulation (43).
Unlike quiescent cells, senescent cells cannot replicate, but instead become enlarged and show multiple molecular changes when stimulated with any physiologic mitogen (44). Senescent cells exhibit enlarged cell volume, an increase in cell surface area, and altered morphology. In the present study, we demonstrated that both stresses induced the chondrocytes to become large and flat. In addition, studies have demonstrated that senescent cells elevate the activity of a unique neutral β-galactosidase, SA–β-gal (26). Because SA–β-gal activation is not related to growth arrest and can be determined in cells in situ, it serves as an ideal biomarker for qualitative and quantitative determination of senescence. Thus, senescence can be measured by activation of SA–β-gal in addition to cell enlargement and inability to replicate. In the present study, we also found that SA–β-gal activity was enhanced in chondrocytes 5 days after a single administration of stress with IL-1β or H2O2. Taken together, these results indicate that both catabolic stresses induce features of chondrocyte senescence.
Erosion beyond the minimum critical telomere length necessary for DNA replication (5–7.6 kbp) results in cell cycle arrest, a phenomenon referred to as replicative senescence (44, 45). Our data showed that stresses with IL-1β and H2O2 accelerated telomere erosion in chondrocytes, and consequently, their lifespans were significantly shortened. These results suggest that in vivo long-term exposure to stress factors may accelerate telomere erosion and induce replicative senescence in arthritic chondrocytes.
Down-regulation of caveolin 1 is sufficient to drive cell transformation to tumorigenicity (46). Previous reports suggest that caveolin 1 may modulate the lifespan of cells (47). Recent work showed that caveolin 1 overexpression arrests mouse embryonic fibroblasts in the G0/G1 phase (48) and that caveolin 1 plays a central role in promoting cellular senescence in fibroblasts (16). Our data showed that both IL-1β and H2O2 up-regulated caveolin 1 mRNA and protein levels. Furthermore, caveolin 1 antisense ODN blocked the features of chondrocyte senescence after stress with IL-1β and H2O2, and caveolin 1 overexpression was able to induce features of chondrocyte senescence. These results indicate that up-regulated expression of caveolin 1 mediates the features of chondrocyte senescence induced by stress with IL-1β and H2O2.
In this context, we also studied the signal transduction pathways involved in features of chondrocyte senescence. It has been reported that p38 MAPK is a senescence-executing molecule that is activated by both telomere-dependent and telomere-independent senescence-inducing stimuli (19), and that the ERK/MAPK pathway is responsible for Ras-induced senescence (20). We found that both IL-1β and H2O2 induced prolonged activation of p38 in chondrocytes. Caveolin 1 overexpression also induced p38 activation. The specific p38 MAPK inhibitor SB202190 blocked features of chondrocyte senescence induced by stress with IL-1β and H2O2, which further supports the notion that prolonged p38 MAPK activation is necessary for stress-induced chondrocyte activity. IL-1β transiently inhibited ERK activation 6–12 hours after stimulation, and H2O2 did not significantly affect ERK activation during the observation period. These findings suggest that ERK activation might not be a common pathway involved in features of chondrocyte senescence accelerated by IL-1β and H2O2. Furthermore, we also found that caveolin 1 overexpression inhibited ERK activation in human articular chondrocytes.
The p53 tumor suppressor protein plays a critical role in regulating cell growth arrest (21); p21 mediates p53-dependent G1 arrest by inhibiting the activity of CDKs, which phosphorylate the Rb gene product, as well as other substrates (22). Unphosphorylated Rb does not release E2F and does not induce G1 entry into the cell cycle (23). In this study, we found that caveolin 1 expression and stresses with both IL-1β and H2O2 up-regulated p53 and p21, but down-regulated phosphorylated Rb in articular chondrocytes. These results suggest that the p53/p21/Rb dephosphorylation pathway may mediate the stress-induced chondrocyte growth arrest.
Although loss of the ability of cells to divide is an accepted measure of senescence, cell function may begin to deteriorate before cell cycle arrest is reached. To demonstrate dysfunction of stress-induced chondrocytes, we confirmed their decreased production of articular cartilage matrix CII and aggrecan. In contrast to the results of previous investigations (3, 30), our studies demonstrated the expression of CII and aggrecan in chondrocytes 3–5 days after stress with IL-1β or H2O2, rather than during incubation with IL-1β or H2O2. An age-related decline in anabolic response to insulin-like growth factor 1 was found in articular cartilage chondrocytes, and chondrocytes from OA cartilage also have a reduced response to insulin-like growth factor 1 (1). These data therefore provide evidence that a cumulative increase of premature senescent chondrocytes in the articular cartilage may provide an alternative explanation for the degeneration of articular cartilage in OA.
In conclusion, our data strongly suggest that in OA, exposure to IL-1β and oxidative stress induces features of chondrocyte senescence, mediated, at least in part, by caveolin 1. Prolonged p38 MAPK activation and the p53/p21/Rb dephosphorylation pathway are necessary to mediate the features of stress-induced senescence. Further studies are needed to clarify whether blocking of caveolin 1 expression prevents cartilage degeneration. Features of chondrocyte senescence accelerated by catabolic factors contribute to the risk of cartilage degeneration by reducing the ability of cells to maintain and repair tissue.