Middlewick, Robert James
(2020)
Investigating a role for heterotrimeric G-proteins in activating TGFβ in asthma.
PhD thesis, University of Nottingham.
Abstract
Asthma is a chronic inflammatory disease of the airways, affecting 5.4 million people in the UK. Asthma pathology is characterised by airway hyper-responsiveness, bronchoconstriction, and airway remodelling. Airway remodelling is particularly prominent in severe asthma and describes structural changes within the airways including, ASM hyperplasia and hypertrophy, subepithelial fibrosis, mucus gland hyperplasia, and angiogenesis. The pleiotropic cytokine TGFβ is plays a central role in the development of all airway remodelling characteristics and is found to have increased activation in asthmatics versus non-asthmatics. Previous studies in ASM cells has shown that the integrin αvβ5 and the actin cytoskeleton is essential for TGFβ activation. Moreover, the heterotrimeric G-proteins Gαq/11 have been implicated in TGFβ activation in murine embryonic fibroblasts and epithelial cells. However, the class of heterotrimeric G-protein required for TGFβ activation in ASM has not been established. The hypothesis of this thesis is that the heterotrimeric G-proteins Gαq/11 are required for TGFβ activation in ASM cells.
The methodology of this thesis uses the GPCR agonist lysophosphatidic acid (LPA) and cyclical mechanical stretch as a stimulus to activate TGFβ in vitro. TGFβ activation has been primarily measured through western blotting of phospho-Smad2 (pSmad2). Inhibiting heterotrimeric G-protein activation has been carried out using a small molecule inhibitor of Gαq/11/14, YM-254890, and siRNA.
The results in this thesis demonstrate, for the first time, that Gαq/11 are not required for either LPA- or stretch-induced TGFβ activation in ASM cells. The serine/threonine kinase, Rho kinase (ROCKI/II), has been shown as central to LPA- and stretch-induced TGFβ activation in ASM cells. Moreover, activation of the small G-protein RhoA has
been shown for the first time to be regulated Gα12/13, but not Gαq/11, in response to LPA in ASM cells. However, inhibition Gα12/13 by siRNA had no effect on LPA-induced pSmad2, therefore implying that LPA-induced TGFβ activation may require ROCKI/II, but not RhoA. Finally, inhibition of both Gαq/11 and Gα12 did not reduce LPA-induced TGFβ activation, however, the role of Gα13 cannot be discounted due to incomplete knockdown.
Collectively, these data disprove the hypothesis that the heterotrimeric G-proteins Gαq/11 are required for TGFβ activation in ASM cells. Moreover, they show that LPA- and stretch-induced TGFβ activation in ASM cells does not require either Gαq/11 or Gα12 signalling. The fascinating possibility that ROCKI/II, but not RhoA, is required for LPA-induced TGFβ in ASM cells requires further investigation. If this hypothesis proves to be true it could unlock new opportunities for drug development to reduce airway remodelling in severe asthma.
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