The influence of intracellular calcium-release in non-canonical TGFB signalling and myofibroblast differentiation

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Ligands of the TGFB superfamily have been implicated in a wide range of physiological processes involving cell growth, migration, survival, differentiation and extracellular matrix production. Consistent with its role in these critical functions, dysregulated TGFB signalling has been associated with diverse disease processes and in aging. In particular the role of TGFB has been highlighted in tissue fibrosis - reported to contribute to around 40% of deaths worldwide annually- where it is recognised as a critical mediator of fibroblast to myofibroblast differentiation and aberrant matrix deposition. Downstream of TGFB receptor activation, canonical signalling occurs via SMAD transcription factors which translocate to the nucleus following receptor mediated phosphorylation to exert their cellular functions. In addition, a number of non-canonical, SMAD-independent pathways have emerged to be critical for TGFB mediated functional responses , including the ERK, PI3K/AKT/mTOR, JNK/p38 pathways and the Rho-like GTPases. Insight into the cross talk between these pathways will be critical in understanding TGFB mediated cellular function in fibrosis and aging.The PI3K/AKT/mTOR axis represents an important oncogenic signalling node, integrating signals from a variety of inputs including tyrosine kinase receptors, G-protein coupled receptors and activated Ras. Recent studies from our laboratory suggest that TGFb induces myofibroblast activation via a novel pathway which utilises mTOR activation independent of PI3K and Akt. The upstream PI3K/Akt independent activator of mTOR is as yet unknown. Transforming growth factor activated kinase (TAK1) has been and implicated in non-canonical TGFB mediated profibrotic gene expression in a variety of in vitro and in vivo models. Importantly recent preliminary transcriptional studies in our laboratory using laser capture microdissection of human biopsy material from the lungs of patients with idiopathic pulmonary fibrosis have highlighted that recognised components of the TAK1 signalling pathway including the calcium dependent phosphatase calcineurin its regulatory partner regulator of calcineurin 1 (RCAN1) and downstream transcription factor nuclear factor of activated T-cells (NFAT) are all highly associated with procollagen type I and III gene expression. Moreover, cytoplasmic levels of Ca2+ are significantly elevated in cells undergoing ER and oxidative stress, conditions prevalent in fibrotic tissue in the lung and other organs. Bringing these streams together, we hypothesise that calcium dependent NFAT activation downstream of TAK1 and mTOR isa critical pathway for myofibroblast differentiation and pro-fibrotic gene expression.In order to test this hypothesis, this studentship will aim to:1) Expand on our preliminary transcriptional findings from laser capture microdissection using additional biopsy tissue from IPF lungs as well as investigating whether the RCAN1/ calcineurin/ NFAT axis correlates with profibrotic gene expression in other organ settings. 2) Define the expression of components of calcineurin signalling and TAK1 complex in our in vitro model systems, using primary fibroblasts isolated from IPF and non-IPF lung tissue as well as primary fibroblasts isolated from other organs. The student will also investigate the presence of NFAT consensus binding sites in extracellular matrix related genes and explore NFAT phosphorylation and translocation to the nucleus following TGFB stimulation3) Access a toolbox of pathway specific inhibitors available within GSK to define the relationship between TAK1 signalling and TGFB mediated mTOR activation in isolated primary fibroblast cultures. Moreover, the student will employ an organotypic lung slice model developed within GSK to pharmacologically explore this pathway in a complex tissue environment.