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Title: The role of connective tissue growth factor (CTGF) in articular cartilage
Author: Tang, Xiaodi
ISNI:       0000 0004 7232 9702
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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Work from our group has identified an important role for the pericellular matrix (PCM) of cartilage in mechanotransduction. This region of the matrix sequesters regulatory molecules such as fibroblastic growth factor 2 (FGF2) and releases them upon mechanical stimulation. By carrying out a proteomic analysis of PCM proteins we identified an additional regulatory molecule, connective tissue growth factor (CTGF). CTGF was of interest to us as this protein is up-regulated in osteoarthritis but its function in chondrocytes is unknown. I confirmed the pericellular localisation of CTGF in cartilage and showed that it binds to heparan sulphate perlecan and like FGF2, CTGF is rapidly released from the PCM upon cartilage injury. In order to determine its function in chondrocytes, I expressed recombinant CTGF and performed a microarray study in isolated human chondrocytes. Four genes were up- regulated by CTGF and all were known to be transforming growth factor beta (TGFβ) inducible. Recombinant CTGF was able to activate SMAD2 in porcine chondrocytes indicating that it can activate the canonical TGFβ signalling pathway. Both CTGF induced gene expression and SMAD2 phosphorylation was abrogated by TGFβ neutralising antibody and the TGFβ receptor inhibitor SB431542, suggesting that the activity of CTGF is TGFβ dependent. Adding exogenous CTGF to rested porcine cartilage increased TGFβ protein accumulation without affecting TGFβ mRNA levels, suggesting that CTGF may control TGFβ protein’s bioavailability. Injured porcine cartilage caused rapid release of endogenous CTGF as well as a delayed accumulation of TGFβ in the conditioned medium (CM). TGFβ proteins are synthesised and secreted in a latent complex associated with the latency- associated peptide (LAP). When I analysed the explantation CM under non-reducing conditions, CTGF resolved at a high molecular weight (~150kDa), and a high molecular weight fraction of this CM contained SMAD2 activity suggesting that CTGF may be in complex with latent TGFβ. This was supported by the identification of LAP in CTGF immunoprecipitates. Injured cartilage from CTGF knockout mice released reduced levels of CTGF but apparently normal levels of TGFβ suggesting that CTGF is not necessary for release of latent TGFβ. However, SMAD2-phosphorylating ability of the medium was compromised indicating that CTGF controls TGFβ bioavailability principally by facilitating activation of latent complex at the surface of the chondrocyte. These data unveil a novel role for CTGF in cartilage through controlling TGFβ bioavailability.
Supervisor: Vincent, Tonia Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral