Aging does not directly cause osteoarthritis (OA); however, it is the most important risk factor in the development of the disease (1). A major contributing factor in the development of OA is a loss of the anabolic and catabolic homeostasis maintained by chondrocytes, leading to a loss of articular cartilage. The balance of anabolic and catabolic processes in cartilage depends on the local activity of regulatory factors such as cytokines and growth factors (2). Insulin-like growth factor 1 (IGF-1) has the ability to stimulate matrix synthesis (3–5), promote chondrocyte survival (6), and inhibit specific catabolic pathways (7, 8). There is evidence that chondrocytes have a decreased response to IGF-1 with aging and also in OA (3, 9–12), but the mechanisms involved are incompletely understood.
IGF-1 stimulates the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway as well as the Ras/Raf/MEK/ERK pathway, by acting through the IGF-1 receptor (13). The activation of the IGF-1 receptor results in the activation of Shc and members of the insulin receptor substrate (IRS) family. After the phosphorylation of IRS and Shc, both the PI 3-kinase cascade and the ERK cascade are activated. The activation of PI 3-kinase leads to the activation of Akt (also called protein kinase B [PKB]), a serine/threonine kinase involved in cell survival (14, 15), and p70 S6 kinase, a serine/threonine kinase implicated in protein synthesis (16).
Activation of PI 3-kinase, but not of ERK, by IGF-1 is required for the stimulation of chondrogenesis and proteoglycan (PG) synthesis by IGF-1 that occurs in mesenchymal cells (17), as well as for the IGF-1–mediated stimulation of PG synthesis that occurs in adult human chondrocytes (18). Although IGF-1 acts as an autocrine survival factor for chondrocytes cultured at low density (6, 16), the required role of PI 3-kinase and/or ERK in IGF-1–mediated cell survival is not clear and may depend on the experimental conditions. Inhibition of the ERK pathway has been reported to reduce survival of chondrocytes plated on collagen (19) but not on fibronectin (20). Inhibition of PI 3-kinase reduced survival of chondrocytes treated with sodium nitroprusside (17) but did not reduce survival of chondrocytes in high-density monolayer or alginate cultures (18).
It has been shown that the tribbles homolog TRB3 (also known as SKIP3, NIPK, or SINK) inhibits IGF-1 as well as insulin activation of Akt/PKB in the liver (21). TRB3 has been classified as a pseudokinase because it lacks the Asp-Phe-Gly motif in subdomain VII of the kinase domain (22). This truncated kinase domain, which lacks the adenosine 5′-triphosphate binding site, allows TRB3 to act as a negative modulator of Akt through dose-dependent inhibition of Akt/PKB phosphorylation at Ser473 and Thr308 in HEK 293 cells (21). TRB3 has also been shown to inhibit the phosphorylation of Akt at Thr308 in FGC-4 cells (23) and to block the insulin activation of p70 S6 kinase, Akt, mammalian target of rapamycin, p70 S6 ribosomal protein, and 4EBP1 in mouse hepatocytes (24). However, a contradictory study in rat hepatocytes could not find evidence that TRB3 inhibits the insulin signaling pathway (25). This discrepancy suggests that the function of TRB3 may be dependent on the cell type and/or experimental conditions.
Investigations into the regulation of TRB3 expression have focused on the role of endoplasmic reticulum (ER) stress. Agents that induce ER stress have been found to increase TRB3 messenger RNA (mRNA) levels in HepG2 cells (26) as well as in p53-null osteosarcoma cells, MCF-7 breast cancer cells, H1299 lung cancer cells, and DU145 prostate cancer cells (27). TRB3 protein levels have also been shown to be increased in conditions of increased ER stress in HepG2, HEK 293, and A375 cells (28). Whether chondrocytes express TRB3 under normal conditions or only under conditions of ER stress has not been examined.
Because of the potential of TRB3 to serve as an inhibitor of IGF-1 signaling, we sought to determine, first, if chondrocytes express TRB3, and then, whether the expression of TRB3 changes with aging or in the presence of OA. We also examined whether an increase in ER stress would increase chondrocyte expression of TRB3, and whether an increased expression of TRB3 might contribute to a decrease in PG synthesis and cell survival. Our results suggest that increased production of TRB3 in OA cartilage may play an important role in the inhibition of chondrocyte IGF-1 signaling.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
This study is the first to show that chondrocytes express TRB3, a protein that has previously been identified in other cell types as an inhibitor of insulin- and IGF-induced activation of Akt/PKB (21, 23, 24). Activation of Akt by PI 3-kinase is required for the IGF-1–mediated stimulation of chondrocyte PG synthesis (18) as well as for cell survival under conditions dependent on autocrine IGF-1 signaling (6). The PI 3-kinase/Akt pathway has also been shown to be required for transforming growth factor β induction of tissue inhibitor of metalloproteinases 3 expression (35). In our study, a significantly greater amount of TRB3 was detected in OA chondrocytes than was detected in age-matched cells from normal-appearing cartilage. Overexpression of TRB3 in normal chondrocytes reduced cell survival and blocked IGF-1 stimulation of PG synthesis. Together, these findings suggest that an increased level of TRB3 in OA chondrocytes could contribute to the decreased response of OA chondrocytes to IGF-1 that has been previously observed (3, 9), and might contribute to the cell death and matrix loss seen during the development of OA.
A potential mechanism for increased expression of TRB3 is ER stress (26–28). Chondrocytes exhibit a variety of behaviors related to different stressors, including osmotic stress, nutrient deprivation, oxidative stress, and mechanical stress (36–38), which can potentially impair ER function, resulting in ER stress (39). Recently, it has been shown that ER stress can be induced in chondrocytes under certain conditions (33, 40, 41). We found that an increase in ER stress induced by either tunicamycin or thapsigargin caused an increase in chondrocyte TRB3 levels. ER stress in chondrocytes has been shown to decrease the expression of cartilage matrix genes (40), and prolonged ER stress leads to apoptosis (33, 41). The present findings demonstrating that overexpression of TRB3 can inhibit IGF-1–mediated PG synthesis and reduce chondrocyte survival suggest a potential mechanism for TRB3 as a mediator of the effects of ER stress in chondrocytes.
TRB3 is a pseudokinase that binds Akt and, thus, prevents the phosphorylation of Akt, a process that is required for Akt to be active (21). The results of our study confirmed that TRB3 blocks the IGF-1–stimulated phosphorylation of Akt in human chondrocytes, similar to previous observations in other cell types (21, 23, 24). Inhibition of Akt activation by TRB3 was associated with reduced chondrocyte survival in low-density, serum-free alginate cultures, an experimental condition in which we have previously demonstrated that IGF-1 autocrine signaling promotes chondrocyte survival (6). This is consistent with other reports showing that TRB3 caused cell death in several different cell types (28, 42, 43).
The IGF-1/PI 3-kinase/Akt pathway is a well-characterized survival pathway in many cell types (14, 15, 44, 45). The PI 3-kinase inhibitor wortmannin was previously shown to decrease the survival of rabbit chondrocytes (46). In the present study, we found that overexpression of a PI 3-kinase dominant-negative construct decreased survival of human chondrocytes. These findings suggest that increased levels of TRB3 cause cell death through inhibition of the IGF-1/PI 3-kinase/Akt survival pathway. When TRB3 was overexpressed in high-density monolayer in medium supplemented with 5% FBS, the negative effects of TRB3 on cell survival could be overcome. Using these conditions, we determined that increased TRB3 levels could inhibit PG synthesis. This is consistent with previous studies in which it was shown that the PI 3-kinase/Akt pathway was required for IGF-1 stimulation of PG synthesis by chondrocytes (18). The finding of increased levels of TRB3 in OA chondrocytes suggests that TRB3 could play a role in the reduced response to IGF-1 that has been noted previously in OA (10). It was less clear whether TRB3 levels were increased with older age in subjects whose cartilage had no OA-related changes; therefore, we do not know, at this time, whether TRB3 also contributes to the age-related decline in the IGF-1 response.
In this study, we were thus able to demonstrate TRB3 production by human chondrocytes and to show that TRB3 levels are dramatically increased in cells from OA cartilage. TRB3 blocks the IGF-1–stimulated phosphorylation of Akt in chondrocytes. Moreover, overexpression of TRB3 causes chondrocyte cell death by inhibiting the PI 3-kinase/Akt pathway in culture conditions in which this pathway is important for chondrocyte survival. Overexpression of TRB3 also inhibits the IGF-1–stimulated PG synthesis by chondrocytes. It was also seen that an increase in ER stress can up-regulate the production of TRB3 in chondrocytes. Together, these results suggest that TRB3 may play an important role in the reduced response to IGF-1 in OA chondrocytes, and therefore TRB3 may contribute to the imbalance in anabolic and catabolic activity that is characteristic of OA. Further studies will be necessary to determine whether inhibition of TRB3 during the development of OA can slow the progression of cartilage loss.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Loeser 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 design. Cravero, Loeser.
Acquisition of data. Cravero, Carlson, Im, Yammani, Long.
Analysis and interpretation of data. Cravero, Carlson, Yammani, Long, Loeser.
Manuscript preparation. Cravero, Carlson, Loeser.
Statistical analysis. Cravero.