Publications
Objective Determining the effect of microRNA-544a-3p (miR-544a-3p) in articular chondrocytes isolated from patients affected by osteoarthritis (OA) and its role in the modulation of the Wnt signalling. Method Articular chondrocytes were isolated from patients undergoing joint replacement because of OA. Expression levels of miR-544a-3p were measured by PCR and in situ hybridization. Putative targets of miR-544a-3p were confirmed by reporter assay and qPCR in cells stimulated with a miR-544a-3p mimic. The effect of miR-544a-3p on chondrocyte metabolism was monitored by qPCR for phenotypic markers, protein expression levels of aggrecan neoepitopes/MMP-13 and modulation of proteoglycan content in micromass cultures, upon stimulation with a miR-544a-3p mimic. The expression levels of MMP-13 and Aggrecan neoepitopes in response to miR-544a-3p stimulation was also measured in co-stimulation with XAV-939 and KN93, respectively β-catenin and CaMKII inhibitors. Results Our results suggest that miR-544a-3p enhances the activation of the Wnt-signalling in articular chondrocytes. The expression of miR-544a-3p is higher in chondrocytes isolated from damaged (median difference Hodges-Lehman, damaged vs preserved, 1.480, 95%CI, 0.1794–2.784, p=0.0097) areas of the articular cartilage removed from OA patients, and can be upregulated by pro-inflammatory cytokines. miR-544a-3p exerts a pro-catabolic effect on articular chondrocytes, which is rescued both by the inhibition of the Wnt/β-catenin and Wnt/CaMKII signalling pathways. Conclusion Our results point to miR-544a-3p as a new, important modulator of the Wnt signalling network within the articular cartilage suggesting a key role for microRNAs in regulating how the multiple branches of the network and their interaction modulate cartilage homeostasis.
Exposure to environmental pollutants has a proven detrimental impact on different aspects of human health. Increasing evidence has linked pollution to the degeneration of tissues in the joints, although through vastly uncharacterised mechanisms. We have previously shown that exposure to hydroquinone (HQ), a benzene metabolite that can be found in motor fuels and cigarette smoke, exacerbates synovial hypertrophy and oxidative stress in the synovium. To further understand the impact of the pollutant on joint health, here we investigated the effect of HQ on the articular cartilage. HQ exposure aggravated cartilage damage in rats in which inflammatory arthritis was induced by injection of Collagen type II. Cell viability, cell phenotypic changes and oxidative stress were quantified in primary bovine articular chondrocytes exposed to HQ in the presence or absence of IL-1β. HQ stimulation downregulated phenotypic markers genes SOX-9 and Col2a1, whereas it upregulated the expression of the catabolic enzymes MMP-3 and ADAMTS5 at the mRNA level. HQ also reduced proteoglycan content and promoted oxidative stress alone and in synergy with IL-1β. Finally, we showed that HQ-degenerative effects were mediated by the activation of the Aryl Hydrocarbon Receptor. Together, our findings describe the harmful effects of HQ on articular cartilage health, providing novel evidence surrounding the toxic mechanisms of environmental pollutants underlying the onset of articular diseases.
Osteoarthritis (OA) is a highly disabling musculoskeletal condition affecting millions of people worldwide. OA is characterised by progressive destruction and irreversible morphological changes of joint tissues and architecture. At molecular level, de-regulation of several pathways contributes to the disruption of tissue homeostasis in the joint. Overactivation of the WNT/β-catenin signalling pathway has been associated with degenerative processes in OA. However, the multiple layers of complexity in the modulation of the signalling and the still insufficient knowledge of the specific molecular drivers of pathogenetic mechanisms have made difficult the pharmacological targeting of this pathway for therapeutic purposes. This review aims to provide an overview of the WNT/β-catenin signalling in OA with a particular focus on its role in the articular cartilage. Pathway components whose targeting showed therapeutic potential will be highlighted and described. A specific section will be dedicated to Lorecivivint, the first inhibitor of the β-catenin-dependent pathway currently in phase III clinical trial as OA-modifying agent.
WNT ligands can activate several signalling cascades of pivotal importance during development and regenerative processes. Their de-regulation has been associated with the onset of different diseases. Here we investigated the role of the WNT/Calcium Calmodulin Kinase II (CaMKII) pathway in osteoarthritis. We identified Heme Oxygenase I (HMOX1) and Sox-9 as specific markers of the WNT/CaMKII signalling in articular chondrocytes through a microarray analysis. We showed that the expression of the activated form of CaMKII, phospho-Ca MKII, was increased in human and murine osteoarthritis and the expression of HMOX1 was accordingly reduced, demonstrating the activation of the pathway during disease progression. To elucidate its function, we administered the CaMKII inhibitor KN93 to mice in which osteoarthritis was induced by resection of the anterior horn of the medial meniscus and of the medial collateral ligament in the knee joint. Pharmacological blockade of CaMKII exacerbated cartilage damage and bone remodelling. Finally, we showed that CaMKII inhibition in articular chondrocytes upregulated the expression of matrix remodelling enzymes alone and in combination with Interleukin 1. These results suggest an important homeostatic role of the WNT/CaMKII signalling in osteoarthritis which could be exploited in the future for therapeutic purposes.