The role of mesenchymal G proteins in pulmonary fibrosis and lung development

Goodwin, Amanda T. (2021) The role of mesenchymal G proteins in pulmonary fibrosis and lung development. PhD thesis, University of Nottingham.

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The maintenance of lung homeostasis is complex and involves the concerted actions of numerous cell types and signalling pathways. Any disturbance to these intricate processes and interactions can result in lung disease, such as pulmonary fibrosis. In pulmonary fibrosis, an abnormal wound healing response involving the abnormal recapitulation of developmental signalling pathways occurs, resulting in scarring of the lungs. An understanding of normal lung developmental pathways, and how they become abnormally reactivated in pulmonary fibrosis, may hold the key to creating regenerative strategies to benefit patients with this condition.

Mesenchymal cells, including fibroblasts, myofibroblasts, and pericytes, play several roles in maintaining lung homeostasis, including the manipulation of extracellular matrix (ECM) properties such as content and mechanical stiffness, cellular crosstalk and transdifferentiation, and transforming growth factor-β (TGFβ) activation. These cells can respond to both chemical and mechanical stimuli, and any disturbance to these external influences can result in an imbalance between lung repair and cellular quiescence, as seen in pulmonary fibrosis. In the developing lung abnormal mesenchymal cell function can halt or disturb normal

developmental processes resulting in structural lung abnormalities, for example as seen in bronchopulmonary dysplasia (BPD). An understanding of how mesenchymal cells detect chemical and mechanical stimuli in lung development and disease could inform the treatment of both pulmonary fibrosis and BPD.

G proteins are essential signalling mediators in numerous physiological processes, mammalian organ development and in the pathogenesis of pulmonary fibrosis. However, the exact roles of these proteins in mesenchymal cell function have not been determined. The aim of this study was to understand the role of the mesenchymal cell G protein families Gαq/11 and Gα12/13 in the pathogenesis of pulmonary fibrosis and provide insight into the manipulation of these signalling mediators as a therapeutic strategy for this condition. This study also aimed to assess the potential adverse effects associated with G protein inhibition in vivo.

Mice lacking mesenchymal Gαq/11 (Pdgfrb-Cre+/-;Gnaqfl/fl;Gna11-/-) from conception had a severely detrimental phenotype, which included growth restriction and abnormal lung appearances consistent with disturbed alveolarisation and reminiscent of BPD. Furthermore, mice lacking mesenchymal Gα12/13 from conception (Pdgfrb-Cre/ERT2;Gna12-/-;Gna13fl/fl) were born abnormally infrequently, suggesting that this genotype resulted in death in utero. While it was hypothesised that mesenchymal cell Gαq/11 or Gα12/13 inhibition may be protective against pulmonary fibrosis, the physical condition of these transgenic mice precluded their use for in vivo pulmonary fibrosis models.

In vitro experiments demonstrated that breathing-related cyclical stretch induced TGFβ signalling in fibroblasts, and this response was elevated in human lung fibroblasts from donors with pulmonary fibrosis. Furthermore, fibrotic human lung fibroblasts were more contractile than non-diseased cells, implying that these cells have greater ECM-organising abilities. Further experiments revealed that Gαq/11, but not Gα12/13, is essential for stretch-mediated TGFβ activation through the generation of TGFβ2. When the response to matrix stiffness was assessed, this study found that Gαq/11 also modulates the myofibroblast phenotype in response to ECM stiffness. Conversely, a contraction assay showed that Gα12/13, but not Gαq/11, is essential for fibroblast contractility at baseline and in response to the G protein coupled receptor (GPCR) agonist lysophosphatidic acid (LPA). Both Gαq/11 and Gα12/13 were found to be important mediators of LPA-induced TGFβ signalling in fibroblasts. As stretch, cellular contraction, ECM properties, and TGFβ signalling are all important in the pathogenesis of pulmonary fibrosis and for normal alveolarisation processes, this study highlights the roles of Gαq/11 and Gα12/13 as shared signalling pathway components between development and disease.

When a tamoxifen-inducible conditional gene knockout model was used mice with mesenchymal Gαq/11 knockdown induced in adulthood developed emphysema. These data indicate that mesenchymal Gαq/11 is responsible for maintaining lung homeostasis, probably via TGFβ signalling, and this represents a shared pathway between pulmonary fibrosis and normal lung development. The breeding of mice with tamoxifen-inducible knockdown of mesenchymal Gα12/13 (Pdgfrb-Cre/ERT2;Gna12-/-;Gna13fl/fl) in adulthood was also found to be feasible. These animals may be suitable for in vivo models of pulmonary fibrosis in future studies.

Overall, the findings of this study demonstrate that mesenchymal cell Gαq/11 and Gα12/13 play essential roles in the pathogenesis of pulmonary fibrosis, normal lung development, and lung homeostasis through responses to mechanical stimuli, cellular contraction, and TGFβ signalling. Further dissection of the processes involved, including the role of specific TGFβ isoforms, could lead to the development of lung regenerative strategies that will benefit patients with a range of respiratory diseases including pulmonary fibrosis and BPD

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Jenkins, Gisli
Johnson, Simon
Keywords: Pulmonary fibrosis; Pathogenesis; Mesenchymal cells; G protein inhibition; Lung homeostasis; TGFβ isoforms
Subjects: W Medicine and related subjects (NLM Classification) > WF Respiratory system
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 65086
Depositing User: Goodwin, Amanda
Date Deposited: 04 Aug 2021 04:41
Last Modified: 04 Aug 2021 04:41

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