Biology of bone, cartilage and connectve tissue disease: 92. WNT3A Modulates Chondrocyte Phenotype through Activation of both Canonical and Non-Canonical Pathways

Abstract

Background: Recent data indicate that Wnt signalling is a major player in the development of osteoarthritis and cartilage breakdown and therefore point to Wnt signalling as an extremely attractive target for osteoarthritis. However some experiments have given contrasting results in the way that both excessive activation and inhibition of this pathway result in increased cartilage breakdown. Wnts can signal through multiple pathways one of which involving the cytoplasmic molecule β-catenin and another one Ca2+ signalling and CaMKII. We have hypothesized that cartilage breakdown following Wnt stimulation is underpinned by a different molecular mechanism of that induced by Wnt inhibition. To test this hypothesis we have blocked at different levels individual pathways mediating Wnt signalling in chondrocytes and have developed a model demonstrating for the first time that one individual Wnt, ligand, Wnt3a, can simultaneously signals through multiple pathways with distinct outcomes and that the individual pathways negatively regulate each other. Methods: Primary articular chondrocytes were isolated from preserved cartilage areas of patients undergoing knee arthroplasty for osteoarthritis. Treatments with Wnt3a alone or in combination with DKK1 (inhibitor of the β-catenin-dependent pathway) or KN93 (CaMKII inhibitor) were performed on confluent primary monolayers or in vivo in the ectopic cartilage formation assay. The activation of the β-catenin dependent pathway was evaluated by reporter assay, nuclear accumulation of β-catenin, or expression of Axin-2. The activation of the Ca2+/ CamKII-dependent pathway was evaluated by Ca2+ mobilization or CamKII phosphorylation (immunofluorescence). Gene expression analysis of proliferation and phenotype markers such as PCNA, COL2A1, Aggrecan and SOX9 was performed by quantitative real time PCR. Results: Wnt3a induced the activation of both the β-catenin-dependent and the CaMKII-dependent pathways. More importantly we have separated the effects that are directly dependent on the activation of the β-catenin-dependent pathway, including proliferation, from those that are CaMKII-dependent such as disruption of the chondrocyte phenotype as evaluated by downregulation of differentiation markers. Most importantly we have built up a model that allows prediction and targeting of individual outcomes of Wnt signalling and explains the paradoxical results of the previous literature. Conclusions: Our findings will allow to therapeutically target individual outcomes of Wnt signalling, for instance to prevent chondrocyte de-differentiation without affecting crucial anabolic processes such as cell proliferation. It will also allow exploition of this extremely potent signalling pathway for therapeutic use in an unprecedented specific manner thereby avoiding potential undesired effects. Disclosure statement: The authors have declared no conflicts of interest.