Agrin mediates chondrocyte homeostasis and requires both LRP4 and α-dystroglycan to enhance cartilage formation in vitro and in vivo

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 a-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 a-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 adystroglycan. 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.