Connective tissue growth factor contributes to joint homeostasis and osteoarthritis severity by controlling the matrix sequestration and activation of latent TGFβ

Abstract

<h3>Objectives</h3> One mechanism by which cartilage responds to mechanical load is by releasing heparin-bound growth factors from the pericellular matrix (PCM). By proteomic analysis of the PCM, we identified connective tissue growth factor (CTGF) and here investigate its function and mechanism of action. <h3>Methods</h3> Recombinant CTGF (rCTGF) was used to stimulate human chondrocytes for microarray analysis. Endogenous CTGF was investigated by in vitro binding assays and confocal microscopy. Its release from cut cartilage (injury CM) was analysed by Western blot under reducing and non-reducing conditions. A postnatal, conditional Ctgf^cKO mouse was generated for cartilage injury experiments and to explore the course of osteoarthritis (OA) by destabilisation of the medial meniscus. siRNA knockdown was performed on isolated human chondrocytes. <h3>Results</h3> The biological responses of rCTGF were TGFβ dependent. CTGF displaced latent TGFβ from cartilage and both were released on cartilage injury. CTGF and latent TGFβ migrated as a single high molecular weight band under non-reducing conditions, suggesting that they were in a covalent (disulfide) complex. This was confirmed by immunoprecipitation. Using Ctgf^cKO mice, CTGF was required for sequestration of latent TGFβ in the matrix and activation of the latent complex at the cell surface through TGFβR3. In vivo deletion of CTGF increased the thickness of the articular cartilage and protected mice from OA. <h3>Conclusions</h3> CTGF is a latent TGFβ binding protein that controls the matrix sequestration and activation of TGFβ in cartilage. Deletion of CTGF in vivo caused a paradoxical increase in Smad2 phosphorylation resulting in thicker cartilage that was protected from OA.