Increased expression of the Akt/PKB inhibitor TRB3 in osteoarthritic chondrocytes inhibits insulin-like growth factor 1-mediated cell survival and proteoglycan synthesis.

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 for the development of OA is a loss of anabolic and catabolic homeostasis 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). 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 age and also in OA (3,9-12), but the mechanisms involved are incompletely understood. IGF-1 stimulates both the 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 IGF-1R 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 ERK cascade are activated. The activation of PI-3 kinase leads to the activation of Akt (also called protein kinase B or PKB), a serine-threonine kinase involved in cell-survival (14,15), and p70S6 kinase, a serine-threonine kinase implicated in protein synthesis (16). IGF-1 activation of PI-3 kinase but not ERK is required for IGF-1 stimulation of chondrogenesis and proteoglycan synthesis by mesenchymal cells (17) and for proteoglycan synthesis by adult human chondrocytes (18). Although IGF-1 acts as an autocrine survival factor for chondrocytes cultured at low density (6,16), the requirement for PI-3 kinase and/or ERK activity for 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 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 and 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 Serine 473 (Ser473) and Threonine 308 (Thr308) in HEK293 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 p70S6Kinase, Akt, mTOR, p70 S6 ribosomal protein and 4EBP1 in mouse hepatocytes (24). However, a contradicting report could not find evidence that TRB3 inhibited the insulin signaling pathway in rat hepatocytes (25). This discrepancy suggests that the function of TRB3 may be dependent on the cell type and/or experimental conditions. Regulation of TRB3 expression has focused on the role of endoplasmic reticulum (ER) stress. Agents which induce ER stress have been found to increase TRB3 RNA levels in HepG2 cells (26) as well as 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 increase with an increase in ER stress in HepG2, HEK 293 and A375 cells (28). Whether or not chondrocytes express TRB3 under normal conditions or conditions of ER stress has not been examined. Because of the potential of TRB3 to serve as an inhibitor of IGF-1 signaling, we examined whether chondrocytes express TRB3 and determined if this expression changes with age or in the presence of OA. We also examined if an increase in ER stress would increase chondrocyte expression of TRB3 and whether an increased expression of TRB3 might contribute to a decrease in proteoglycan synthesis and cell survival. The results suggest that increased TRB3 in OA cartilage may play an important role in inhibiting chondrocyte IGF-1 signaling.