Eley, Jessica; (2018) Investigating the role of mTOR signalling in mediating the pro-fibrotic effects of TGF-β1. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Tissue fibrosis, for which there are at present limited therapeutic strategies, is characterised by abnormal wound healing responses resulting in excessive extracellular matrix (ECM) production, leading to significant tissue architecture distortion and impaired organ function. In the lung, fibrosis may present as idiopathic pulmonary fibrosis (IPF), a chronic, progressive and ultimately fatal condition with unknown aetiology and poor prognosis. The key cytokine mediating progression of IPF and other fibrotic conditions is transforming growth factor beta 1 (TGF-β1), driving fibroblast to myofibroblast differentiation, defined by de novo expression of alpha smooth muscle actin (α-SMA) stress fibres, and stimulating the synthesis and secretion of ECM proteins including collagen I.To examine TGF-β1-induced collagen I deposition by primary human lung fibroblasts (HLFs), I have optimised a high-content assay that facilitates extracellular conversion of soluble procollagen monomers into insoluble collagen I fibrils, a process that is inefficient in traditional 2-dimensional fibroblast culture. This study utilised the assay alongside potent, highly-selective pharmacological compounds to identify a critical role for mTOR signalling in the extracellular deposition of collagen I and de novo induction of α-SMA. Furthermore, this study delineated the role of specific mTOR signalling substrates and defined a key role for 4E-BP1 mediated cap-dependent mRNA translation in the regulation of TGF-β1-dependent, rapamycin insensitive de novo protein synthesis to modulate collagen I and α-SMA synthesis at the mRNA level in HLFs. The potential for mTOR activation as a core signalling node in the pro-fibrotic response to TGF-β1 in effector cells of other tissues has also been explored, and a critical role for this axis in the regulation of α-SMA and collagen I synthesis has been identified in primary human dermal fibroblasts and hepatic stellate cells. Overall, ATP-competitive inhibition of mTOR and its downstream signalling may therefore represent a novel therapeutic strategy in fibrotic disease across multiple organs.

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