Redding, Matthew; (2020) Investigating the pathways required for TGF-β₁ stimulated mTORC1 activation and collagen I synthesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung scarring disease. The abnormal and aberrant wound healing response leads to excess matrix production. The aetiology of IPF is unknown, the prognosis is poor and current treatments are ineffective. Transforming growth factor-β is a critical cytokine which is increased in IPF patients, due to repetitive epithelial injury. TGF-β1 is responsible for driving the progression of the disease through the activation and differentiation of local and recruited fibroblast populations. TGF-β1 initiates signalling pathways within the fibroblasts which drives their differentiation programme into the myofibroblast and the deposition of matrix proteins, in particular collagen I. TGF-β1 regulates genes involved in fibrogenesis. It does this by regulating several transcription factors as well as promoting the translation of the mRNA. Critically, it mediates the transcription of the collagen I gene through the activation of SMAD 3 and the translation of this gene via mammalian target of rapamycin complex 1 (mTORC1) activation. The mechanism of TGF-β1 stimulated mTORC1 activation is currently unknown in primary human lung fibroblasts. To examine the TGF-β1 stimulated pathway required for mTORC1 activation I employed pharmacological, siRNA and gene editing techniques. This study defined the mechanisms of mTOR phosphorylation at the amino acid residue site S2448 and ruled it was not required for TGF-β1 mediated collagen synthesis. This study delineated that ras homology enriched in brain (RHEB) was critical for mTORC1 activation. However, the role of RHEBs regulatory complex, comprised of tuberous sclerosis 1 and tuberous sclerosis 2 (TSC1/2), still remains ambiguous. Finally, it identified that mothers against decapentaplegic (SMAD) 3 was required for early mTORC1 activation through an unknown transcriptionally regulated protein. The work reported in this thesis raises the possibility that there is a protein acting as a link between SMAD 3 and the mTORC1 regulatory components TSC1/2 and RHEB which could be a potential drug target for the treatment of IPF. There could be additional benefits of inhibiting this target rather than directly inhibiting TGF-β1, the TGF-β1 receptors or mTORC1 which have unwanted and serious side-effects. The niche targeting of a SMAD 3 transcriptionally regulated protein that enhances mTORC1 signalling downstream of TGF-β1 should in theory minimise these unwanted side-effects. Finally, the implications of SMAD 3 and mTORC1 activation downstream of TGF-β1 are relevant in other fibrotic diseases adding to the potential of a translatable drug between diseases.

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