Article Text
Abstract
Objectives 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.
Methods 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 CtgfcKO 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.
Results 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 CtgfcKO 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.
Conclusions 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.
- osteoarthritis
- arthritis
- chondrocytes
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Footnotes
Handling editor Josef S Smolen
Contributors TLV and JS devised the project. XT, HM, CM, JM-Z, JF, AD and PÖ generated experimental data. AL developed the CTGF floxed mouse. TLV and XT wrote the manuscript. All authors critically reviewed the manuscript and approved the final version.
Funding This project was funded by the Kennedy Trust for Rheumatology Research who supported studentships for XT and CM (grant no. MSP 10/11/08). The project was also supported by the Arthritis Research UK Centre for OA Pathogenesis (grant no. 20205).
Competing interests AL is a shareholder of FibroGen.
Patient consent Not required.
Ethics approval All animal experiments were carried out with full ethical approval in accordance with local and national regulations.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement Mainly full data sets are presented. Further information is available on request to corresponding author.