Wnt16 is Induced by Oxidative Stress Following Cartilage Injury and Contributes to Cartilage Homeostasis in Osteoarthritis

Purpose: The purpose of this study is to investigate the role of Wnt16 following cartilage injury and osteoarthritis development. Our previous studies showed that Wnt16 is up-regulated upon acute trauma -a condition known to predispose to osteoarthritis (OA) - in an ex vivo setting. Activation of Wnt signalling is associated with cartilage breakdown and OA development, although the mechanisms and the ligands responsible for it are still unclear. The aims of this study, therefore, are i) to investigate whether Wnt16 contributes to the outcome of experimental OA in vivo; ii) The molecular mechanism leading to WNT16 up-regulation following injury and iii) identify which signalling pathway is activated by Wnt16 in vivo. Methods: Articular chondrocytes were isolated from bovine metatarsal joints. The cells were then stimulated with recombinant Wnt16 at different doses and gene expression analysis performed. OA was induced by destabilization of the medial meniscus (DMM) in 8 week-old 129/Sv mice Wnt16-/- or WT littermate. The animals were culled at 2, 7 days and 8 weeks post surgery. The joints were either processed for gene expression analysis, fixed for bone analysis (μCt) or de-calcified and paraffin embedded, for histological, morphometric, and immunohistochemical characterization. The degree of cartilage breakdown was assessed by using OARSI score by two independent investigators. Six animals/genotype were left ageing for 1 year to evaluate the development of spontaneous OA. To test the involvement to oxidative stress in Wnt16 modulation, organ cultures of articular cartilage explanted from femoral heads were treated with H2O2 in presence/absence of a NADPH oxidase inhibitor. Gene expression analysis for Wnt16 and cartilage markers performed afterwards. Results: Wnt16 expression was upregulated at 2 and 7 days post-DMM and was associated with an increased expression of β-catenin. Modulation of the Wnt-βcatenin pathway was confirmed in vitro on bovine articular chondrocytes stimulated with recombinant Wnt16. Wnt16-deficient mice developed more severe cartilage breakdown in comparison to WT littermate following DMM, whereas, if left unchallenged, did not develop spontaneous OA with age. Wnt16-deficient mice displayed a lower expression of lubricin, the absence of which is known to result in OA. Finally, we found that mechanical trauma to cartilage resulted in oxygen radical release, and this was required and sufficient to trigger WNT16 upregulation in chondrocytes. Indeed H2O2 alone was sufficient to up-regulate WNT16, whereas chemical blockade of NAPDH oxidase inhibited WNT16 up-regulation induced by mechanical trauma. Conclusions: We demonstrated for the first time that Wnt16 supports cartilage homeostasis following injury, and it is required to reduce degradation upon mechanical trauma. We also showed that oxidative stress, previously thought to play a pathogenic role in cartilage breakdown in the first phases of OA development, in our settings, drives Wnt16 up-regulation, suggesting a new homeostatic role for oxidative stress. Finally, we show that Wnt16 supports the expression of lubricin. Our results shed new light on the role of Wnt signalling in the articular cartilage and open a new window of opportunity to modulate the Wnt pathway for the treatment of acute and chronic cartilage injury.