Agrin inhibits canonical WNT signalling through LRP4

Abstract

Purpose: 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. We recently identified Agrin as an essential molecule in articular cartilage homeostasis, uniquely requiring both α-dystroglycan and LRP4 to support SOX9 expression and chondrocyte differentiation in vitro and in vivo. Despite identifying α-dystroglycan and LRP4 as necessary for the chondrogenic effects of Agrin in chondrocytes, the signalling pathway(s) by which this occurs remains unclear. LRP4 signalling is known to negatively regulate WNT signalling. Therefore, we tested whether Agrin is required for the WNT-inhibitory properties of LRP4 and whether this is the mechanism by which Agrin signals in chondrocytes. Methods: Mammalian Agrin or GFP was overexpressed in bovine primary chondrocytes and cultured in micromass for 5 days in the presence or absence of 100ng/ml WNT3a. Chondrogenic potential was assessed by qPCR for SOX9 mRNA expression. Agrin or GFP stably expressing COS7 cells were transfected with the SUPER8TOPFLASH reporter plasmid in combination with Vehicle or LRP4 plasmids. COS7 monolayer cultures were treated with vehicle or increasing doses of WNT3a (50, 100, 200ng/ml). WNT signalling activation was determined by luciferase expression. Results: LRP4 overexpression was sufficient to induce SOX9 upregulation in chondrocytes, however this effect was abolished following Agrin knockdown demonstrating the requirement of Agrin for the chondrogenic effect of LRP4 signalling. In addition, downregulation of SOX9 mRNA expression induced by WNT3a treatment was rescued by Agrin overexpression in micromass cultures. Using the WNT reporter assay, activation of canonical WNT signalling was inhibited in COS7 cells transduced with human Agrin compared to GFP-transduced control COS7 cells; and this effect was exacerbated by overexpression of LRP4. Conclusions: Agrin inhibits canonical WNT signalling through LRP4 and enhance chondrocyte differentiation. However, it remains unclear how Agrin-LRP4 signalling achieves its specificity in promoting chondrogenesis compared to other LRP4 ligands.