Spatiotemporal regulation of anti-remodelling signalling pathways in primary human lung fibroblasts

Roberts, Maxine (2020) Spatiotemporal regulation of anti-remodelling signalling pathways in primary human lung fibroblasts. PhD thesis, University of Nottingham.

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Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disease caused by a dysregulated wound healing process that results in fibrosis and scarring of lung tissue rather than repair. Current approved IPF therapeutics target multiple known fibrotic mediators and pathways, however this multi target-kinase approach results in numerous severe adverse effects. Dysregulated lung fibroblasts are one of the key cell types involved in the progression of lung fibrosis. Increasing evidence has revealed the role of cAMP in inhibiting fibroblast pro-fibrotic phenotypic responses via crosstalk with the mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK) pathway. However, what makes cAMP elevating agonists efficacious at inhibiting pro-fibrotic process is unclear.

By activating Gs-coupled G protein-coupled receptor (GPCRs) that we showed to be expressed in human lung fibroblasts, we confirm that increasing cAMP inhibits fibroblast proliferation and differentiation. However, the efficacy of agonists to generate cAMP did not correlate to its ability to inhibit phenotypic responses. This disconnect was observed when targeting the prostacyclin receptor (IPR) with a partial agonist for generating cAMP (MRE-269) having more efficacy at inhibiting fibroblast proliferation in comparison to full agonists for generating cAMP (iloprost/treprostinil). We hypothesised that the location of cAMP accumulation and/or the temporal regulation of cAMP signalling may be important for the inhibition of fibroblast proliferation.

As such, targeted fluorescence resonance energy-transfer (FRET) biosensors were used to investigate cAMP and ERK signalling with high spatial and temporal resolution. A complex relationship was discovered between IPR cAMP elevating agents and the inhibition of ERK in different cellular compartments. We demonstrated that IPR agonists that are efficacious at inhibiting fibroblast proliferation have sustained nuclear cAMP activity and can inhibit nuclear ERK. This suggests nuclear cAMP and the inhibition of nuclear ERK may be important for the inhibition of phenotypic responses. However, forskolin, which increases cAMP independent of receptor activation and is efficacious at inhibiting fibroblast proliferation, had a sustained nuclear cAMP response but was unable to inhibit nuclear ERK.

GloSensorâ„¢ cAMP assay was used to measure cAMP levels at a range of time points. We demonstrated that IPR agonists that are low efficacy agonists at acute time points, maintained efficacy at later time points, whereas high efficacy agonists had a reduction in efficacy. By comparing efficacy of IPR agonists to increase cAMP at acute time points vs later, more phenotypically relevant time points, we demonstrated that efficacy switched, with partial agonists switching to full agonists and full agonists switching to partial agonists.

The activation of IPR signalling, using select IPR agonists, represents a potential therapeutic approach for the treatment of IPF. This study demonstrated the importance of measuring cAMP spatially and temporally to understand agonist actions. Although spatial control of cAMP may have some role in phenotypic response more research needs to be conducted. The most significant result was the reversal of efficacy at phenotypically relevant readouts, demonstrating that sustained cAMP signalling may be important for IPF therapeutics.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Charlton, Steven
Rosethorne, Elizabeth
Keywords: cAMP elevating agents; Novel therapeutic agents; Lung fibroblasts; Idiopathic pulmonary fibrosis
Subjects: R Medicine > RC Internal medicine
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 59568
Depositing User: Roberts, Maxine
Date Deposited: 17 Jul 2020 04:40
Last Modified: 17 Jul 2022 04:30

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