MMP3 cleaves agrin in cartilage and reduces its chondrogenic potency

Abstract

Purpose: Osteoarthritis is a chronic disabling disease characterized by cartilage breakdown for which there is no cure. We recently discovered that the heparan sulphate proteoglycan Agrin is expressed in cartilage, where it is essential for the maintenance of the chondrocytic phenotype and for the production of cartilage extracellular matrix whilst exogenous Agrin enhances chondrocyte differentiation and cartilage formation in vitro and in vivo. Here we study the mechanism that leads to loss of Agrin in OA and the functional consequences of Agrin cleavage by metalloproteinase 3 (MMP3). Methods: Agrin expression was determined by immunohistochemistry. Osteoarthritis was induced in 8 week old 129sv mice by destabilisation of the medial meniscus (DMM) and N-Agrin and C-Agrin expression was evaluated by immunofluorescence at 7 days and 8 weeks post-surgery. Ex vivo human cartilage samples were treated with MMP3 and the cleavage of C-Agrin was compared by immunofluorescence. The effect of MMP3 on Agrin transfected chondrocytes was analysed by qPCR. Results: We discovered that detection of Agrin in osteoarthritic samples is lost in earlier stages of disease progression when using an antibody detecting the C-terminal portion of Agrin compared to when using an antibody recognizing the N-terminal domain. This could be explained by the well-described MMP3 mediated Agrin cleavage which separates the N-terminal domain (predicted to adhere to the basement membrane through interaction with laminin) from the C-terminal domain. In keeping with this hypothesis, treatment of chondrocyte cultures or cartilage explants with recombinant MMP3 resulted in the loss of Agrin detection when using the C-terminal, but not the N-terminal antibody. Using antibodies specific to the N and C-terminal portions of Agrin, we found that C-Agrin is lost in the articular cartilage as early as 2days post surgical-induction of OA (destabilization of the medial meniscus), whereas N-Agrin remains highly detectable for at least 7days post surgical-induction of OA. In functional terms, treatment with recombinant MMP3 reduced the capacity of Agrin to upregulate SOX9 mRNA. Conversely, overexpression of Agrin in chondrocytes strongly downregulated MMP3 mRNA expression, thereby suggesting a negative feedback loop. Conclusions: The cleavage of Agrin by MMP-3 in chondrocytes may contribute to the progression of OA by reducing SOX9 transcription.