Identification of signaling pathways mediating human chondrocyte degeneration induced by the chemokine CCL-2

Abstract

Purpose: Increased expression of chemokines in cartilage, synovial membrane and subchondral bone are believed to be linked to osteoarthritis (OA). The chemokine receptor CCR2 and its major human circulating ligand CCL2 (a.k.a. MCP-1), have been recognized as important potential targets in OA and have been shown also to mediate OA pain. In previous clinical studies, we determined that CCL2 serum levels at baseline were significantly associated with radiographic knee OA progression and joint space narrowing at 5-year follow up. Our findings in a murine model of OA based on the destabilization of the medial meniscus (DMM), showed that the articular cartilage of OA knees had increased protein levels of the chemokine CCL12, a murine CCL2 homologue that binds the same CCR2 receptor, while it was undetectable in controls; CCL12 protein levels increased progressively with OA severity. Interestingly, systemic blockage of the CCR2 at early OA stages, led to decreased levels of matrix metalloproteinases (MMPs) in articular cartilage, 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 μM) 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), following short or long CCL-2 treatments.