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Title: An investigation of the actions of connective tissue growth factor on human renal epithelial cells
Author: Winn, Simon
ISNI:       0000 0004 7427 141X
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2018
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Introduction. Connective tissue growth factor (CTGF) is the only CCN member recognized as a mediator of chronic fibrotic disease. Accumulating evidence suggests that CTGF is important as a downstream effector of transforming growth factor (TGFβ) in modulating a sustained pro-fibrotic signal in in vivo models. Moreover, in cultured human proximal tubule epithelial cells (PTECs) CTGF mRNA is readily upregulated by TGFP and the nascent protein accumulates extracellularly. How pro-fibrotic signalling occurs is yet to be ascertained but it likely involves extracellular interactions with secreted CTGF. The tetra-modular structure of CTGF has been shown to possess multiple binding sites for ligands present in the extracellular milieu including matrix proteins, growth factors including the TGFβ superfamily and cell surface receptors. As such, CTGF can behave as a bridge between matrix and cell. Moreover, individual modules possess intrinsic biologic activity. Materials and Methods. Experiments were performed in cultured renal human renal epithelial cells. Recombinant human CTGF protein was generated in-house following plasmid transfection into context relevant PTECs. Immunoblotting and ELISA techniques were used to investigate protein expression in response to incubation with study protein in isolation or in combination with TGFP superfamily members. Protein-protein binding was investigated using surface plasmon resonance (SPR) in order to elucidate how CTGF might regulate TGFβ superfamily cell signalling. Additional experiments with an alternative CCN member, CCN3, were performed. Results. Both full-length and C-terminal CTGF bind to TGFβ and BMP7 and in human renal epithelial cells, this binding modulates the downstream Smad signalling pathways associated with fibrosis. In cultured human podocytes, CTGF drives TGFβ-dependent signalling in the absence of exogenous TGF(3 suggesting activation of latent TGFβ. Moreover, C-terminal CTGF increases the generation of pro-fibrotic markers aSMA and fibronectin, both of which are subsequently blocked by inhibiting the TGFp receptor. Unlike CTGF, an alternative CCN member CCN3 reduces TGFβ-induced signalling in PTECs. Conclusion. CTGF protein has multiple binding sites and modulates the cellular responses of TGFp superfamily members on human renal epithelial cells. Both full-length and C-terminal CTGF bind to TGFβ and BMP-7. CTGF also appears to bind to the receptors of the TGFβ superfamily. C-terminal CTGF has intrinsic profibrotic activity that differs from the full-length protein as demonstrated in cultured human podocytes. The pro-fibrotic activity is suppressed by CCN3 in the CTGF rich environment of the cultured PTEC. The interplay of different CCN proteins in modulating fibrotic pathways in renal epithelial cells warrants further investigation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available