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Hall, Robert (2020) Functional genetics of lung function associated gene GPR126. PhD thesis, University of Nottingham.

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Abstract

GPR126 lies on chromosome 6q24 and variants in this region are reproducibly associated with lung function and COPD in genome wide association studies (GWAS).This association signal includes a non-synonymous single nucleotide polymorphism (SNP), rs17280293 (minor allele frequency = ~2.9%) which results in the amino acid substitution Ser123Gly within the GPR126 coding region. GPR126 is a G protein coupled receptor belonging to the adhesion family of receptors. The aim of the work described in this thesis was to define the role ofGPR126 in the human lung and in pulmonary disease. My specific aims were to i) identify the GPR126 expression profile in the lung and in airway structural cells ii) determine potential causal variants within the association signal iii) define the effects of missense variant Ser123Gly on GPR126 function iv) determine the function of GPR126 signalling in human airway structural cells.

Linkage disequilibrium (LD) and expression quantitative trait loci (eQTL) analyses were used to determine potential causal variants within the 6q24 association signal. GPR126 mRNA expression in the lung,human bronchial epithelial cells (HBEC) and human airway smooth muscle cells (HASM)was established using qPCR and RNA-seq. GPR126 protein expression was characterised in lung tissue using immunohistochemistry.A recombinant CRE-luciferase reporter cell line was engineered to express the Ser123 and Gly123 variants of GPR126. These cell lines were treated with potential GPR126 agonists and cAMP driven gene expression was measured by luciferase activity. These agonists included the stachel peptide which is a short amino acid sequence in the N-terminal region of the receptor which can activate receptor signalling. Column chromatography was used to measure GPR126 driven cAMP accumulation in HBECs and HASMs in response to activation with the stachel peptide. To assess the downstream effects of GPR126 activation in human airway cells, HASMs were activated with the stachel peptide in the presence and absence of PKA inhibitor, H89,to identify the PKA dependency of this response. Differential gene expression was analysed by RNA-seq. Pathway analysis was carried out with GSEA from the Broad Institute.

GPR126 mRNA is expressed inthe lung, HBEC and HASMs and GPR126 protein expression was detected in human lung. The stachel peptide activates GPR126 in a recombinant cell system, although this signal was not genotype-dependent. Moreover, the stachel peptide also activates endogenous GPR126 in HASMs but not HBECs.

GPR126 activation of HASMs resulted in 514 and 520 differentially expressed genes at 4 and 24 hours post-treatment respectively (5% FDR). There was approximately 20% (61 upregulated and 121 downregulated) overlap between these two time points. This sustained response was partially cAMP-PKA dependent with ~40% of genes no longer differentially expressed with H89 pre-treatment at both time points. Genes upregulated by GPR126 activation are involved in inflammatory pathways (IL33 and ADAM8) and downregulated genes include CTGF and SERPINE1,which both have known roles in fibrosis. Gene set enrichment analysis revealed that beta defensin pathways were upregulated by GPR126 activation where pathways involved in cancer, cell cycle checkpoints and cell proliferation were downregulated.

GPR126 expression was identified in the human lung and airway structural cells. While a potentially causal variant was identified in Ser123Gly no potential effects of this mutation on GPR126 signalling were observed. GPR126 signals in human airway smooth muscle cells and the downstream effects of GPR126 activation were assessed by RNA-seq. HASMs respond to GPR126 agonist and this response has partial PKA dependency implicating other pathways driving gene expression differences we observed. Taken together, this work describes novel biology for GPR126 implicating a role for GPR 126 in regulating airway inflammation and airway remodelling.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Sayers, Ian Hall, Ian P.
Keywords:	GPR126; Pulmonary disease; Gene expression profile; Missense variant Ser123Gly' Cell signalling
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:	59498
Depositing User:	Hall, Robert
Date Deposited:	17 Jul 2020 04:40
Last Modified:	17 Jul 2020 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/59498

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