Gyftaki Venieri, Dafni Ariadni;
(2022)
Investigating the role of YAP and mechanosensing for the identification of new drug targets in skin fibrosis.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Stiffening of the skin is an important driver of fibrogenesis caused by activation of quiescent tissue resident fibroblasts into myofibroblasts. Fibroblasts respond to changes in their extracellular matrix (mechanosensing) leading to altered intracellular signalling. The Yes-associated protein (YAP) is the main effector of the HIPPO mechanosensing pathway, known to negatively regulate woundhealing to avoid excessive fibrotic responses through YAP phosphorylation and cytoplasmic retention or degradation. Under fibrotic responses, YAP phosphorylation leads to nuclear localisation mediating expression of fibrotic genes, adding to the pathogenesis, propagation or maintenance of fibrotic diseases like scleroderma. This thesis explores YAP behaviour in primary skin fibroblasts seeded on hydrogels of different stiffnesses, treated with TGFβ or HIPPO and other mechanosensing pathway inhibitors. It identifies anti-fibrotic drug targets that prevent YAP nuclear localisation after creating a YAP-responsive luciferaseexpressing cell line and testing libraries of potential YAP inhibitors in a highthroughput assay. Results indicate that YAP nuclear localisation is primarily affected by mechanosensing, while TGFβ signalling only propagates these responses. A high throughput assay on the YAP-responsive cell line identified two compounds that significantly decreased YAP/TEAD binding in skin fibroblasts (Dasatinib and PD173955), both of which directly affect SRC/c-ABL kinases. YAP expression after knockdown of the literature-identified NUAK Family Kinase 1 (NUAK1) was also significantly decreased. Treatment of fibroblasts with NUAK1 inhibitors (HTH01-015 and WZ 4003) decreased staining of YAP, while treatment with the SRC/cABL inhibitors decreased both NUAK1 and YAP staining and affected actin filament architecture. TGFβ is a known regulator of SRC/c-ABL kinases and it is also shown to increase NUAK1 staining. In summary, TGFβ stimulation of skin fibroblasts on stiff surfaces, leads to activation of SRC, increased NUAK1 expression and YAP nuclear localisation, providing an alternative pathway for fibrosis.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Investigating the role of YAP and mechanosensing for the identification of new drug targets in skin fibrosis |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10149276 |
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