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accompanied by reduced joint damage and pain. The aim of the present study was to analyze downstream mediators of CCL2/CCR2 signaling in human articular chondrocytes that lead to the activation of genes involved in cartilage degradation. Methods: We used human articular chondrocytes from normal cartilage obtained from tissue donors to determine the signaling pathways activated by CCL2 (through CCR2) that lead to production of MMPs. Human chondrocyte primary cultures were treated with or without recombinant CCL2 (20ng/ml) in serum free medium for 5, 10 and 15 minutes. Cell lysates were collected and subjected to Immunoblotting (IB) using antibodies against phospho-ERK, phospho-p38 and phospho-JNK. To determine whether CCL2 treatments led to changes in the expression of genes involved in cartilage degeneration, human articular chondrocytes with or without recombinant CCL2 were incubated in serum free medium for mRNA collection; two different incubations were performed, a short treatment (6h) and a long treatment (18-24h). Quantitative RT-PCR analyses were performed to quantify the gene expression of mmp-1, mmp-3, mmp-13, comp and timp-1 using TaqMan method. Gene expression for the ccr2 receptor was also assessed. In some experiments, human articular chondrocytes were pre-incubated with the ERK inhibitor U0126 (10 mM) for 1 hour prior to CCL2 treatment, and then IB (for phospho-Erk) or qRT-PCR analyses (for mmp-13 and mmp-3) were performed. Results: CCL2 treatments of human chondrocytes led to activation of phospho-ERK and phospho-p38, but not phospho-JNK. When gene expression was assessed by RT-PCR, both short (6h) and long (18-24h) CCL2 treatments led to an upregulation of ccr2 expression compared to untreated controls, indicating a CCL2 positive feedback on its receptor. Both short and long CCL2 treatments also led to an upregulation in the expression of mmp's genes (mmp-1, mmp-3, and mmp-13), as well as an increased expression of the tissue inhibitor of metalloproteinases-1 (timp-1) and comp, which has been demonstrated to be a biomarker for cartilage breakdown. Inhibition of ERK signaling by U0126 abolished the CCL-2-induced upregulation of mmp-13, at both the short and long incubation times ( Figure 1). Interestingly, inhibition of ERK signaling did not significantly affect mmp-3 upregulation by CCL-2. Conclusions: Our data suggest that CCL2 stimulation of articular cartilage chondrocytes acts as positive feedback on its CCR2 receptor leading to cartilage degradation. The triggering of the CCL2/CCR2 axis leads to the activation of specific MAPK signaling pathways, such as ERK and p38 signaling. In particular, our results suggest that ERK signaling mediates the CCL-2-induced mmp-13 activation but may not be involved, or only partially involved, in the mmp-3 upregulation. Additional studies are currently in progress in our laboratory to determine whether inhibition of ERK and p38 is correlated to the expression of different genes involved in cartilage metabolism and degradation (such as mmp-1, timp-1, comp), fol...

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Purpose: Healthy cartilage homeostasis depends on an intact collagen scaffold and high aggrecan content. ADAMTS-5 (A Disintegrin And Metalloprotease with ThromboSpondin-motifs-5) is critically involved in arthritic diseases because of its direct role in cleaving aggrecan. Several studies indicate that inhibition of ADAMTS-5 may have the potential to stop progression of osteoarthritis (OA). In the present study, we investigated the in vitro efficacy of M6495, an inhibitor of ADAMTS-5, including assessment of affinity, potency, specificity, and its effect on the levels of glycosaminoglycan (GAG) and the ADAMTS-5 generated neoepitope of aggrecan (huARGS) in bovine and human cartilage explant assays. Furthermore, we studied the effect of M6495 on cartilage derived markers from aggrecan (GAG) and type II collagen (C2M), as well as type III collagen (C3M) in a cartilage-synovium co-culture model. Methods: Binding kinetics of M6495: The affinity of M6495 for human ADAMTS-5 was determined via Sapidyne's 'in solution' affinity platform Kinetic Exclusion Assay (KinExA). To determine the binding region of M6495 within ADAMTS-5, binding experiments were performed using SPR (Surface plasmon resonance). Inhibition of enzymatic activity of ADAMTS-5 by M6495 was analyzed with a fluorescence resonance energy transfer (FRET)-based enzymatic activity assay. The specificity of M6495 towards ADAMTS-5 was assessed by binding experiments to homologous metalloproteinases. Explant assay and co-culture: The effect of M6495 (half maximal inhibitory concentration [IC 50 ]) in bovine and human cartilage explants stimulated with pro-inflammatory cytokines was determined. Biomarkers of cartilage turnover, including GAG and huARGS, were investigated in the supernatant after culture had ended. Bovine cartilage explants and synovial membranes were coincubated. GAG as a measure for aggrecan turnover, C2M as a marker for MMP (matrix metalloproteinase) mediated type II collagen degradation and C3M as a marker for MMP-mediated type III collagen degradation were analyzed in the supernatant of the cultures. Results: M6495 is a bifunctional Nanobody® of 28.1 kDa (i.e., one ADAMTS-5-neutralizing moiety and one [HSA]-binding moiety for in vivo half-life extension). M6495 binds ADAMTS-5, but not ADAMTS-1, ADAMTS-4 and ADAMTS-15. In-vitro binding studies revealed that M6495 binds to the catalytic and/or disintegrin domain of ADAMTS-5 with high affinity. Results from FRET assays indicate a concentrationdependent and complete inhibition of the enzymatic activity of ADAMTS-5 by M6495. M6495 dose-dependently inhibited GAG release in bovine-, and GAG and huARGS release in human cartilage explant assays. In the co-culture model, matrix degradation was induced in the presence of synovium. First aggrecan was degraded (GAG release) followed by type II collagen degradation (C2M release). M6495 inhibited both. In addition, C3M was determined in the supernatant of the coculture system. Our data revealed a significant induction of C3M levels in co-cultures compared to t...

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