Activation and disruption of Wnt/β-catenin signaling both result in cartilage breakdown via unknown mechanisms. Here we show that both WNT-3A and the Wnt inhibitor DKK1 induced de-differentiation of human articular chondrocytes through simultaneous activation of β-catenin–dependent and independent responses. WNT-3A activates both the β-catenin–dependent canonical pathway and the Ca2+/CaMKII noncanonical pathways, with distinct transcriptional targets. WNT-3A promotes cell proliferation and loss of expression of the chondrocyte markers COL2A1, Aggrecan, and SOX9; however, proliferation and AXIN2 up-regulation are downstream of the canonical pathway and are rescued by DKK1, whereas the loss of differentiation markers is CaMKII dependent. Finally, we showed that in chondrocytes, the Ca2+/CaMKII-dependent and β-catenin–dependent pathways are reciprocally inhibitory, thereby explaining why DKK1 can induce loss of differentiation through de-repression of the CaMKII pathway. We propose a novel model in which a single WNT can simultaneously activate different pathways with distinct and independent outcomes and with reciprocal regulation. This offers an opportunity for selective pharmacological targeting.

Objective Both excessive and insufficient activation of WNT signalling results in cartilage breakdown and osteoarthritis. WNT16 is upregulated in the articular cartilage following injury and in osteoarthritis. Here, we investigate the function of WNT16 in osteoarthritis and the downstream molecular mechanisms. Methods Osteoarthritis was induced by destabilisation of the medial meniscus in wild-type and WNT16-deficient mice. Molecular mechanisms and downstream effects were studied in vitro and in vivo in primary cartilage progenitor cells and primary chondrocytes. The pathway downstream of WNT16 was studied in primary chondrocytes and using the axis duplication assay in Xenopus. Results WNT16-deficient mice developed more severe osteoarthritis with reduced expression of lubricin and increased chondrocyte apoptosis. WNT16 supported the phenotype of cartilage superficial-zone progenitor cells and lubricin expression. Increased osteoarthritis in WNT16-deficient mice was associated with excessive activation of canonical WNT signalling. In vitro, high doses of WNT16 weakly activated canonical WNT signalling, but, in co-stimulation experiments, WNT16 reduced the capacity of WNT3a to activate the canonical WNT pathway. In vivo, WNT16 rescued the WNT8-induced primary axis duplication in Xenopus embryos. Conclusions In osteoarthritis, WNT16 maintains a balanced canonical WNT signalling and prevents detrimental excessive activation, thereby supporting the homeostasis of progenitor cells.

Objectives Osteoarthritis (OA) is a leading cause of disability for which there is no cure. The identification of molecules supporting cartilage homeostasis and regeneration is therefore a major pursuit in musculoskeletal medicine. Agrin is a heparan sulfate proteoglycan which, through binding to low-density lipoprotein receptor-related protein 4 (LRP4), is required for neuromuscular synapse formation. In other tissues, it connects the cytoskeleton to the basement membrane through binding to α-dystroglycan. Prompted by an unexpected expression pattern, we investigated the role and receptor usage of agrin in cartilage. Methods Agrin expression pattern was investigated in human osteoarthritic cartilage and following destabilisation of the medial meniscus in mice. Extracellular matrix (ECM) formation and chondrocyte differentiation was studied in gain and loss of function experiments in vitro in three-dimensional cultures and gain of function in vivo, using an ectopic cartilage formation assay in nude mice. Receptor usage was investigated by disrupting LRP4 and α-dystroglycan by siRNA and blocking antibodies respectively. Results Agrin was detected in normal cartilage but was progressively lost in OA. In vitro, agrin knockdown resulted in reduced glycosaminoglycan content, downregulation of the cartilage transcription factor SOX9 and other cartilage-specific ECM molecules. Conversely, exogenous agrin supported cartilage differentiation in vitro and ectopic cartilage formation in vivo. In the context of cartilage differentiation, agrin used an unusual receptor repertoire requiring both LRP4 and α-dystroglycan. Conclusions We have discovered that agrin strongly promotes chondrocyte differentiation and cartilage formation in vivo. Our results identify agrin as a novel potent anabolic growth factor with strong therapeutic potential in cartilage regeneration.

ROR2 blockade improves the functional and structural outcomes of osteoarthritis in preclinical animal models by inhibiting YAP signaling.