Objective. S100 proteins have been implicated in various inflammatory conditions, including arthritis. The aims of this study were to determine whether chondrocytes produce S100A4 and whether S100A4 can stimulate the production of matrix metalloproteinase 13 (MMP-13) by articular chondrocytes via receptor for advanced glycation end products (RAGE)-mediated signaling.Methods. The expression of chondrocyte S100A4 was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage and by immunoblotting of chondrocyte cell lysates. RAGE signaling was examined by stimulating chondrocytes with S100A4 and monitoring for the activation of MAP kinases and NF-B. Production of MMP-13 was determined in the conditioned medium. A pulldown assay using biotinlabeled S100A4 was used to demonstrate binding to RAGE.Results. S100A4 expression was detected in human articular chondrocytes by immunoblotting and appeared to increase in the cell lysates from OA tissue. Marked positive immunostaining for S100A4 was also noted in sections of human cartilage with changes due to OA. Stimulation of chondrocytes with S100A4 increased the phosphorylation of Pyk-2, MAP kinases, and activated NF-B, followed by increased production of MMP-13 in the conditioned medium. This signaling was inhibited in cells pretreated with soluble RAGE, advanced glycation end product-bovine serum albumin, or the antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin, or by overexpression of a dominant-negative RAGE construct. A pulldown assay showed that S100A4 binds to RAGE in chondrocytes.Conclusion. This is the first study to demonstrate that S100A4 binds to RAGE and stimulates a RAGEmediated signaling cascade, leading to increased production of MMP-13. Since both S100A4 and RAGE are up-regulated in OA cartilage, this signaling pathway could contribute to cartilage degradation in OA.

Objective. The receptor for advanced glycation end products (RAGE) binds multiple ligands, including S100 proteins, high mobility group box chromosomal protein 1 (HMGB-1), and AGEs, all of which are present in articular cartilage. Stimulation of RAGE signaling can lead to MAP kinase activation and increased NF-B activity. The objective of the present study was to determine if chondrocytes express functional RAGE.Methods. The presence of chondrocyte RAGE was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage from young and old monkeys and humans, immunoblotting of chondrocyte lysates and human cartilage extracts, and reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA from chondrocytes treated with interleukin-1 (IL-1) and fibronectin fragments. RAGE signaling was evaluated by stimulating chondrocytes with S100B and HMGB-1 and analyzing for activation of the ERK MAP kinase and NF-B. The ability of S100B and HMGB-1 to stimulate matrix metalloproteinase 13 (MMP-13) production was also assessed. A pull-down assay using biotin-labeled S100B was used to demonstrate binding to RAGE.Results. RAGE was detected in sections of monkey knee cartilage and human knee and ankle cartilage. Increased immunostaining for RAGE was noted in cartilage from older adult monkeys and humans and was further increased in OA tissue. RAGE was also detected by immunoblotting and by RT-PCR, where IL-1␤ and fibronectin fragments were found to stimulate RAGE expression. Stimulation of chondrocytes with S100B or HMGB-1 increased phosphorylation of the ERK MAP kinase and the p65 subunit of NF-B and increased the production of MMP-13. This signaling was inhibited in cells pretreated with soluble RAGE, and S100B was shown to bind to chondrocyte RAGE.Conclusion. Articular chondrocytes express functional RAGE. The increase in RAGE noted in OA cartilage and the ability of RAGE ligands to stimulate chondrocyte MAP kinase and NF-B activity and to stimulate MMP-13 production suggests that chondrocyte RAGE signaling could play a role in OA.

Objective. The chondrocyte response to insulinlike growth factor 1 (IGF-1) is reduced with aging and in osteoarthritis (OA). IGF-1 signals through the phosphatidylinositol 3-kinase/Akt pathway. TRB3, a tribbles homolog, has been shown to inhibit IGF-1-mediated activation of Akt in HEK 293 cells. This study was undertaken to determine if TRB3 is expressed in chondrocytes, and whether the chondrocyte response to IGF-1 is reduced by TRB3.Methods. Human articular cartilage was obtained from normal tissue donors and from patients with OA at the time of knee replacement surgery. TRB3 was assessed in the tissue samples by reverse transcriptionpolymerase chain reaction, immunoblotting, and immunohistochemistry. Overexpression of TRB3 was induced by transient transfection to determine the effects of TRB3 on cell survival and proteoglycan synthesis.Results. TRB3 messenger RNA was detected in normal human chondrocytes. TRB3 protein levels were low in cells from normal cartilage but significantly increased in cells from OA cartilage. Incubation with 2 agents that induce endoplasmic reticulum stress, tunicamycin and thapsigargin, increased TRB3 levels in normal cells. Overexpression of TRB3 inhibited Akt phosphorylation and reduced chondrocyte survival and proteoglycan synthesis.Conclusion. These results are the first to demonstrate that TRB3 is present in human chondrocytes, and that the level of TRB3 is increased in OA cartilage and in isolated OA chondrocytes. Because it is an inhibitor of Akt activation, elevated TRB3 production could play a role in the increased cell death and reduced response to IGF-1 observed in OA cartilage.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 Supported by NIH grant AG-16697.

S100 proteins are low molecular weight calcium binding proteins expressed in vertebrates. The family constitutes 21 known members that are expressed in several tissues and cell types and play a major role in various cellular functions. Uniquely, members of the S100 family have both intracellular and extracellular functions. Several members of the S100 family (S100A1, S100A2, S100A4, S1008, S100A9, S100A11, and S100B) have been identified in human articular cartilage, and their expression is upregulated in diseased tissue. These S100 proteins elicit a catabolic signaling pathway via receptor for advanced glycation end products (RAGE) in cartilage and may promote progression of arthritis. This review summarizes our current understanding of the role of S100 proteins in cartilage biology and in the development of arthritis.

Cubilin, a 456 kDa multipurpose receptor lacking in both transmembrane and cytoplasmic domains is expressed in the apical BBMs (brush border membranes) of polarized epithelia. Cubilin interacts with two transmembrane proteins, AMN, a 45-50 kDa protein product of the amnionless gene, and megalin, a 600 kDa giant endocytic receptor. In vitro, three fragments of cubilin, the 113-residue N-terminus and CUB domains 12-17 and 22-27, demonstrated Ca2+-dependent binding to megalin. Immunoprecipitation and immunoblotting studies using detergent extracts of rat kidney BBMs revealed that cubilin interacts with both megalin and AMN. Ligand (intrinsic factor-cobalamin)-affinity chromatography showed that in renal BBMs, functional cubilin exists as a complex with both AMN and megalin. Cubilin and AMN levels were reduced by 80% and 55-60% respectively in total membranes and BBMs obtained from kidney of megalin antibody-producing rabbits. Immunohistochemical analysis and turnover studies for cubilin in megalin or AMN gene-silenced opossum kidney cells showed a significant reduction (85-90%) in cubilin staining and a 2-fold decrease in its half-life. Taken together, these results indicate that three distinct regions of cubilin bind to megalin and its interactions with both megalin and AMN are essential for its intracellular stability.