Functional genomics of GPR126 in airway smooth muscle and bronchial epithelial cells

GPR126 is an adhesion G protein‐coupled receptor which lies on chromosome 6q24. Genetic variants in this region are reproducibly associated with lung function and COPD in genome wide association studies (GWAS). The aims of this study were to define the role of GPR126 in the human lung and in pulmonary disease and identify possible casual variants. Online tools (GTEx and LDlink) identified SNPs which may have effects on GPR126 function/ expression, including missense variant Ser123Gly and an intronic variant that shows eQTL effects on GPR126 expression. GPR126 signaling via cAMP‐mediated pathways was identified in human structural airway cells when activated with the tethered agonist, stachel. RNA‐seq was used to identify downstream genes/ pathways affected by stachel‐mediated GPR126 activation in human airway smooth muscle cells. We identified ~350 differentially expressed genes at 4 and 24 hours post stimulation with ~20% overlap. We identified that genes regulated by GPR126 activation include IL33, CTGF, and SERPINE1, which already have known roles in lung biology. Pathways altered by GPR126 included those involved in cell cycle progression and cell proliferation. Here, we suggest a role for GPR126 in airway remodeling.

genetic component with recent heritability estimates ranging from 50% to 66%. 3 However, the mechanisms underlying genetic effects are mostly unclear.
Lung function measurements are essential parameters used in the diagnosis of COPD and are, therefore, ideal quantitative traits to use in studies aiming to identify the genetic risk factors for COPD. The most commonly used are forced expired volume in 1 second (FEV 1 ), forced vital capacity (FVC), and FEV 1 /FVC ratio. Using lung function phenotypes for genetic studies allows for additional subjects and, therefore, increased power to detect causal variants with the assumption that the genetics of lung function and COPD share some commonality. The most recent and largest genome wide association study (GWAS) for lung function identified 279 loci showing association with FEV 1 , FVC, or FEV 1 /FVC. 4 Moreover, GWAS in COPD subjects have identified that there is significant overlap between signals associated with COPD risk and signals associated with lung function measurements. 5 There is now a need to translate GWAS signals to biological understanding by studying the underlying functional effects of the genetic variants in respiratory health and disease.
Moreover, missense variant Ser123Gly (signal 1) is also associated with diffusing capacity of the lung (D LCO /V A ) (risk allele A, beta = −0.07, and P value = 1.51E-10) even after adjustment for FEV 1 /FVC. 8 This study also noted that GPR126 expression was decreased in lung tissue obtained from more severe COPD patients compared with never smokers and in patients with lowered D LCO /V A compared to normal D LCO /V A controls. 8 GPR126 belongs to the adhesion GPCRs (aGPCRs) subfamily of GPCRs. This family has 33 members, defined by the presence of their large N-terminal regions, which can be as large as 6000 amino acids long, in addition to the canonical 7 transmembrane spanning regions (7TM). 9,10 These extracellular regions contain several conserved domains that are important for adhesion interactions. All aG-PCRs contain the GPCR autoproteolysis-inducing (GAIN) domain which contains the GPCR proteolysis site (GPS) motif. 11,12 During biosynthesis, aGPCRs are autoproteolysed at this site which results in a cleaved receptor that nonetheless remains associated at the cell surface. 13,14 While the extracellular domain (ECD) is still associated with the 7TM it exerts an inhibitory effect on receptor signaling and it is hypothesized that interaction with a potential ligand or mechanical stimuli would result in removal or rearrangement of the ECD thereby removing the inhibitory effect. 15,16 This reorganisation releases a short peptide sequence which lies C-terminal to the GPS cleavage site (termed stachel). This stachel peptide acts as a tethered agonist and can activate receptor signaling. 17 These receptors can signal via traditional GPCR pathways via G proteins and 18 to date Gα12/13, Gαo, Gαs, Gαi, and Gαq have all been shown to be involved in aGPCR signaling pathways. 15 GPR126 is one of the more well-studied aGPCRs with known roles in peripheral nerve development, angiogenesis, and inner ear formation via canonical GPCR signaling through G as -and G ai -mediated regulation of intracellular cAMP. [19][20][21][22][23][24][25] Complete knockout of GPR126 in mice leads to embryonic lethality due to cardiovascular failure. 26 However, incomplete knockout where the ECD is still present leads to mice which have limb defects, highlighting an independent signaling role for the ECD once dissociated from the 7TM. 23,26,27 A number of potential ligands have been identified for GPR126 including laminin-211 and collagen type IV, which are predicted to involve binding to the complement C1r/C1s, Uegf, Bmp1 (CUB), and pentraxin (PTX) domains in the ECD. 23,28 Ser123Gly (signal 1) lies within the CUB domain and, therefore, could have potential effects on ligand binding. Moreover, recent studies have identified additional mechanisms which modulate GPR126 activity, including the importance of a calcium-binding domain in the ECD, mutations in which lead to defects in myelination and ear development in vivo. 29 Furthermore, alternative splicing of exon 6 (present in 2/4 known isoforms) modulates GPR126 signaling activity. 29 Isoforms lacking exon 6 maintain a closed conformation and show decreased signaling activity compared to those with exon 6, which inhibits the closed conformation. 29 Polymorphisms in GPR126 have been associated with scoliosis in humans. 18,[30][31][32][33] One such variant, rs41289839, has been shown to decrease the expression of exon 6, which would be expected to dampen GPR126 signaling activity. 18,29 Despite advances in understanding of GPR126 function, the role of GPR126 in the human lung is currently unknown and, therefore, the mechanisms by which genetic variants at the GPR126 locus are associated with lung function are not understood. However, it is known that GPR126 is differentially expressed throughout human lung development, suggesting that it may be important for normal lung development. 34

HALL et AL.
We hypothesized that GPR126 is the causal gene underlying the genetic associations at this locus and that either alteration in protein structure or regulation of the levels of the mRNA/protein underlie this effect. In this study, we aimed to identify potential causal variants within GPR126 and identify potential mechanisms by which altered GPR126 expression/ function may affect airway biology.

| RNA isolation
RNA isolation was carried out using the GenElute™ Mammalian Total RNA Miniprep Kit as per the manufacturer's instruction (Sigma).

| cDNA synthesis
cDNA synthesis was performed using the Superscript First Strand Synthesis System for RT-PCT kit (Invitrogen).

| Human airway smooth muscle cell culture
Human lung tissue was obtained from the Nottingham Health Science Biobank or the Papworth Hospital Tissue Bank from patients undergoing lung resection surgery. Human airway smooth muscle (HASM) cells were obtained from lung tissue using the explant model outlined below. Details of these tissue donors can be found in the supplement (Supplementary Table 2). Written consent was obtained from all patients and the study was conducted under the approval of the research Tissue Banks (ethics reference 10/H1008/72 and 08/ H0304/56+5).
Small sections of airways were dissected from the surrounding tissue and the resulting explants were smeared across the bottom of one well of a 6-well plate. Cells were grown out from explants cultured in DMEM supplemented with 10% of FCS and 5% of PSF.

| Recombinant cell line production
GloResponse™ CRE-luc2P HEK293 cells were transfected using Fugene® 6 (Promega) with a lentivirus plasmid containing full length GPR126 and the puromycin resistance gene at a 1.5:1 Fugene: DNA ratio using the protocol was provided by the manufacturer. Cells expressing GPR126 were selected by puromycin treatment. Plasmids were designed and purchased from VectorBuilder (Chicago, USA).

| Luciferase assays
Transfected cells were seeded into 96-well plate 24 hours prior to stimulation and treated with the stachel peptide (1 mM) or other agonists for 4 hours. Bright-Glo buffer and Bright-Glo substrate (Promega) were mixed and 100 µl of the solution was added to each well of a 96-well plate to terminate the assay. Plates were incubated at room temperature for at least 2 minutes before luminescence was measured on a Flexstation 3 microplate reader (Molecular Devices) for 0.5 seconds/well.

| cAMP assays
cAMP responses were measured as previously described. 36 In brief, HBEC and HASM cultures were seeded into 24-well plate 24 hours prior to treatments. Cell cultures were loaded with 3 H adenine for 2 hours. Cells were washed with serumfree media prior to stimulation. Experiments were stopped and cells were lysed by adding concentrated HCl. 14 C-cAMP was added to each well to allow for normalization across the columns. The contents of each well were then transferred to Dowex columns and three milliliters of H 2 O was added to wash each column. Dowex columns were placed on top of alumina columns. Six milliliters of H 2 O was added to dowex columns and allowed to drip through both sets of columns. Dowex columns were removed and the alumina columns were placed over scintillation vials. Five milliliters of 0.1 M of imidazole was applied and allowed to drip through the columns into the scintillation vials. Ten milliliters of scintillation fluid was added to each vial and vials were counted on a Packard Scintillation counter using a 3 H/ 14 C dual-count program for 5 minutes per vial. cAMP accumulation was calculated by normalizing 3 H counts per well to 14 C counts. Each condition was performed in three replicate wells.

| Library preparation for RNAsequencing
Library preparation and RNA-sequencing were carried out by Oxford Genomics Centre. RNA was quantified using RiboGreen (Invitrogen, Glasgow, UK) on the FLUOstar OPTIMA plate reader (BMG Labtech, Aylesbury, UK) and the size profile and integrity analyzed on the 4200 TapeStation (Agilent). RNA was normalized to 100 ng prior to library preparation. Polyadenylated transcript enrichment and strand-specific library preparation were completed using NEBNext Ultra II mRNA kit (NEB, Hitchin, UK) following the manufacturer's instructions. Libraries were amplified on a Tetrad (Bio-Rad, London, UK) using in-house unique dual-indexing primers. Individual libraries were normalized using Qubit, and the size profile was analyzed on the 4200 TapeStation. Individual libraries were normalized and pooled together accordingly. The pooled library was diluted to ~10 nM for storage. The 10 nM library was denatured and further diluted prior to loading on the sequencer.

| RNA-sequencing
Paired end (75 bp) sequencing was performed using a HiSeq4000 platform and HiSeq 3000/4000 PE Cluster Kit and 150 cycle SBS Kit (Illumina, Cambridge, UK), generating a raw read count of ~30 million reads per sample.

| Analysis of RNA-sequencing data
Processing of the RNA-sequencing data was performed using a Unix operating system. Adaptor sequence and quality trimming were performed using Scythe (https://github.com/vsbuf falo/scythe) and Sickle (https://github.com/najos hi/sickle), respectively. Alignment of RNA-Seq reads to genome build GRCh37 was carried out using Tophat package. 37 Analysis of trimmed RNA-sequencing files was performed using the Cufflinks package 37 as we have done previously. 38 Cuffdiff and Cuffnorm were used to determine differential (5% FDR) and gene expression was normalized to library size, respectively. Gene set enrichment analysis was performed using the GSEA software from the Broad Institute and the Molecular Signatures Database (MSigDB). 39,40 Volcano plots and heatmaps were produced in RStudio using ggplot2 41 and pheatmap, 42 respectively.

| Variants on chromosome 6 associated with lung function alter GPR126 amino acid sequence and affect GPR126 expression levels
To identify causal variants underlying the two association signals with FEV 1 /FVC ratio, we sought to identify SNPs which could be potentially causative. This included identifying SNPs which have regulatory effects such as expression quantitative trait loci (eQTLs) and non-synonymous variants using GTEx Portal V8 Release (https://www.gtexp ortal.org/ home/) 43 and Open Target Genetics (https://genet ics.opent argets.org/). We also used LDlink and HaploReg V4.1 to identify additional variants that were in linkage disequilibrium (LD) with the two sentinel variants (r 2 > 0.1).
Signal 1 (rs17280293) results in missense variant Ser123Gly. Using LDlink, we identified 64 SNPs in LD with signal 1 (r 2 > 0.1) including missense variant rs11155242 ((Lys230Gln), r 2 = 0.1442, D' = 1). Neither SNP is an eQTL for GPR126 expression in lung tissue in the GTEx database (V8 release). However rs17280293 is an eQTL for GPR126 expression in blood (eQTLGen database) 44 with the G allele (higher lung function) associated with reduced GPR126 expression (beta: −0.205, pval:7.1e-16). In addition, rs11155242 (C, correlated with rs17280293 G) is an eQTL for GPR126 in a number of tissues including blood, macrophage, monocytes, and T-Cells. 45 SIFT 46 and PolyPhen-2 47 were used to assess potential pathogenicity of these two coding region variants. Both tools predicted rs17280293 to be damaging to protein function potentially via the insertion of a smaller amino acid and loss of structure and rs11155242 to be benign/tolerated. Signal 2 had 262 SNPs in LD (r 2 > 0.1) and is in linkage equilibrium with rs17280293 (r 2 : 0.0836) suggesting independence from signal 1. Within this LD block there are two SNPs associated with scoliosis rs41289839 (r 2 = 0.1746) and rs6570507 (r 2 = 0.9006) with rs7753012. The signal 2 sentinel SNP (rs7753012) is an eQTL for GPR126 expression in the esophagus with the G allele (higher lung function) associated with lower expression of GPR126 (NES: -0.15, pval: 0.000031). Moreover, the two scoliosis SNPs are also eQTLs for GPR126 in other tissues including blood, cultured fibroblasts, and skeletal muscle. 43

| GPR126 is expressed in human lung tissue and structural airway cells
To determine the expression profile of GPR126 in human structural airway cells, we performed RNA-sequencing on RNA extracted from HASM cells and HBECs. HBECs were from healthy donors purchased from Lonza (Wokingham, UK) and HASM cells were grown from lung tissue donors.
In order to assess if GPR126 expression in human lung tissue varies in disease states such as COPD, we utilized RNA-seq datasets available on the Gene Expression Omnibus (GEO). We searched the database with the following search terms; Lung tissue, airway smooth muscle, bronchial epithelial, COPD, and RNA-seq. We identified two datasets which allow comparison of gene expression in lung tissue/cell types between COPD and control.
GSE57148 contains a dataset comparing lung tissue from patients with normal spirometry vs tissue from COPD patients. 48 GPR126 expression is expressed in human lung tissue and at significantly higher levels in COPD patients vs controls (COPD FPKM median 10.5, control FPKM median 9.1, and P = .0164) ( Figure 1B). GSE12 4180 contains RNAseq data from large airway epithelium, alveolar macrophage, and peripheral blood samples from COPD cases and controls. 49 Differential expression of GPR126 was not observed in large airway epithelium, alveolar macrophages, or peripheral blood between controls and COPD patients, although sample numbers were low ( Figure 1C-E).

| Adhesion GPCR expression in structural airway cells
To contextualize the expression levels of GPR126, we analyzed RNA-seq data in HASM and HBECs for the wider F I G U R E 1 Average FPKM values for all donors is shown for HBECs (n = 9) and HASMs (n = 5) (A). RNA-seq data from whole lung tissue was downloaded from GEO (GSE57148) and GPR126 expression was compared in RNA extracted from healthy lung tissue and lung tissue from COPD patients (B). FPKM values from control (mean = 9.1, min = 2.1, and max=18.4) and COPD patients (mean = 10.1, min = 1.7, and max=22.5) were compared using an unpaired t test. Data were normally distributed as determined by a Shapiro-Wilk normality test. GPR126 expression in COPD vs controls was compared in GSE12 4180 in different cell types (C-E). No significant differences in expression between COPD and control were observed family of aGPCRs as well as the expression levels of a wellstudied airway GPCR for comparison, the β-2 adrenergic receptor (ADRB2) ( Table 1). Seventeen of the 33 aGPCRs are expressed in at least one cell type and GPR126 is expressed in both HASM and HBEC at higher levels than ADRB2.

| GPR126 signals via cAMP-mediated pathways in structural airway cells
In order to assess GPR126 signaling activity and effects of the Ser123Gly variant to potential ligands, we produced a recombinant cell system using the GloResponse™ CRE-luc2P HEK293 Cell Line (Promega) to overexpress human GPR126. We created cells lines expressing the Ser123 and Gly123 variants of the receptor. Cells were treated with collagen type IV (3 µg/ml) and laminin-211 (200 ng/µl) for 4 hours; however neither agonist induced signaling. 23,28 Cells were then treated with the activating peptide stachel (1 mM) for 5 hours 17 and we observed a dose-dependent increase in luciferase activity in cells expressing both variants but no increase in signal in empty vector or no vector controls ( Figure 2A). However, we did not identify any genotypespecific effects. We also measured cAMP accumulation in these same cells using column chromatography, showing increased levels of cAMP after 1 mM stachel treatment at 1 and 4 hours ( Figure 2B).
We then tested whether stachel could activate GPR126 signaling in primary cultured human airway structural cells. HASM cells and HBECs were treated with stachel peptide (1 mM) for 1 hour with pre-incubation with IBMX (10 uM). Accumulation of cAMP was measured using column chromatography. In HASM cells we observed a mean fold increase in cAMP of 21.5 ± 3.4 (P = .0079) and in HBECs a more modest but still significant response (2.1 ± 0.5 mean fold increase (P = .0278)) ( Figure 2C-D).

| GPR126 expression increases as cells become more confluent and after stachel stimulation
These data show that activation of cAMP-driven gene expression occurs after stimulation of GPR126 expressing cells with stachel peptide. Therefore, to gain insight into potential biological functions driven by changes in gene expression in HASM and identify downstream targets of GPR126 activation, we treated HASM cells with stachel (1 mM) for 4 and 24 hours and performed RNA-sequencing. Cuffnorm was used to generate normalized expression values (FPKM) for individual genes and isoforms in HASM cells (n = 4 independent experiments, one donor). GPR126 is expressed at higher levels 24 hours after treatment and is significantly elevated after stachel treatment for 24 hours compared to basal and scrambled (P = .0071) (Figure 3). There was no significant difference in GPR126 expression between the different conditions after 4 hours.

| GPR126 activation results in differential regulation of >300 genes
To assess the downstream genes and pathways and, therefore, potential biological implications of GPR126 activation, we used Cuffdiff to assess differentially regulated genes between HASM cells treated with stachel and those treated with a scrambled control peptide.

| Genes implicated in airway diseases are regulated by GPR126 activation
After identifying genes which were differentially regulated at the two time points, we compared the genes differentially expressed at 4 hours to those at 24 hours. We found that ~20% of genes were differentially regulated by GPR126 activation with the same direction of effect with 40 upregulated and 74 downregulated ( Figure 6A). To focus our analysis, we then looked at the genes which were differentially expressed using an arbitrary cut off of a ≥2 fold change in either direction at both time points ( Figure  6B). There were 26 genes that met this criteria, these include genes with previously reported roles in airway disease such as IL33 (upregulated), CTGF (downregulated), and SERPINE1 (downregulated). 50-52

| Pathway analyses suggests GPR126 may have a role in cell proliferation
Gene Set Enrichment Analysis (GSEA) was used from the Broad Institute to identify gene sets/biological pathways effected by GPR126 activation. 40 There were nine gene sets that were enriched (1% FDR) by GPR126 activation in the hallmark gene sets. No gene sets were enriched at 4 hours using the hallmark gene set. The differentially regulated gene sets at 24 hours are involved in cell cycle regulation and cell proliferation ( Table 2). These processes are known to be F I G U R E 2 Luciferase response of HEK293 cells transfected with GPR126 to increasing concentrations (100-1000µM) of the stachel peptide after 5 hours of incubation (n = 5) (A). cAMP measurements in GPR126 Ser123 and GPR126 Gly123 HEK293 cells in response to stachel peptide, isoprenaline, and forskolin treatment for 10, 60, 120, and 240 minutes with pre-treatment with IBMX (B). cAMP accumulation in HASM cells (C) and HBECs (D) pre-treated with IBMX and then, treated with Stachel (1 mM) or isoprenaline (10 µM) for 1 hour (n = 5). Data shown are mean ± SEM. SFM, serum free media regulated by aGPCRs in other cell types. We also analyzed the list of differentiated genes (1% FDR) through The Database for Annotation, Visualization, and Integrated Discovery (DAVID). 53 The most significant results grouped differentially expressed genes into Mitosis (P = 2.23E-06), cell division (P = 1.54E-03), and cell cycle (P = 9.11E-03).

| DISCUSSION
In this study, we hypothesized that genetic variants that affect GPR126 expression or function may contribute to processes that lead to airway disease. We aimed to identify potential causal variants driving the GWAS association signals observed for FEV 1/ FVC and explore the potential role of GPR126 in human airway structural cells. Signal 1 has a coding region sentinel SNP resulting in Ser123Gly, a variant predicted to be damaging to protein function, but in recombinant cell experiments we were unable to detect any altered signaling as a consequence of this polymorphism. The signal 2 top variant however is an eQTL in a range of tissue types where it has been studied, suggesting that altered GPR126 expression could be driving at least some of the genetic associations seen. We identified GPR126 expression in the lung and in HASMs and HBECs and identified that GPR126 could have a role in airway remodeling via transcriptomic analysis.
From the analysis of the LD structure around the sentinel SNPs, the most compelling SNP for further analysis was the Ser123Gly variant (signal 1), predicted to be damaging to protein function. Based on the genetics from the Shrine et al, 2019 GWAS, the Ser123 allele (common) is protective against lung function decline.
However, we were unable to detect a functional effect of the Ser123Gly variant in recombinant cell models. There are several potential explanations for this. First, stachel binds Cterminal to the Ser123Gly position and, therefore, any potential effects of this amino acid change on ligand binding may be bypassed. We were unable to test any differential effects of the reported ligands collagen type IV and laminin 211 as these did not activate receptor signaling in our system. We cannot exclude that the recombinant expression system (higher than endogenous) altered the state of GPR126 and responsiveness to ligand. Second, we overexpressed the alpha 2 variant of the receptor, which lacks exon 6 of the full-length protein and this may, therefore, be a limitation. It has recently been shown that transcripts lacking exon 6 exist in a closed conformation and signal less readily than GPR126 transcripts with exon 6 present. 18 These factors may explain why signaling activity was not observed when cells were treated with collagen type IV or laminin 211. However, we saw robust effects on signaling in both cell lines and also in primary cultured HASM cells following activation with the stachel peptide suggesting any genotype-specific effects should have been found with the transcript we expressed.
Our eQTL analyses suggest that alleles associated with higher lung function are associated with reduced GPR126 mRNA expression levels. Similarly, we identified elevated GPR126 mRNA levels in lung tissue from COPD patients vs normal spirometry controls. Therefore, we hypothesis that increased expression/activity of GPR126 may be damaging in the lung. We chose to study this in airway smooth muscle, given that eQTL signals were observed in fibroblast and smooth muscle containing tissues.
Given the potential for altered gene expression to be driving effects of GPR126 signaling, we therefore, used RNAseq to identify the potential roles of GPR126 in HASMs by activating the receptor with the activating peptide stachel. GPR126 activation by stachel peptide resulted in 345 and 356 differentially expressed genes at 4 and 24 hours, respectively. There were distinct transcriptome profiles at the two time points with a ~20% overlap of common differentially regulated genes. Therefore, while the two time points may identify early and late response genes, there is increased confidence that the genes differentially regulated at both time points are driven by real effects of GPR126 activation. The genes differentially regulated following the activation of GPR126 are predominantly in pathways controlling mitosis and cell division. We also found that GPR126 mRNA expression in HASMs generally increases over time in culture. One potential explanation for the increase in GPR126 expression at 24 hours may be that the cells were more confluent and, therefore, there are more cell-cell and cell-matrix interactions. As the cells proliferate, more matrix is deposited and an increase in matrix would be expected to include matrix ligands for GPR126. aGPCRs are known to modulate a number of cellular functions including cell migration, adhesion, polarity, and guidance via these adhesion interactions. 54 Moreover, it Genes which were not differentially regulated in three out of four replicates were removed. Brown represents upregulation and green represents downregulation is also known that GPR126 is vital for normal tissue development via interactions with the ECM. 23,28 Therefore, GPR126 may be upregulated to facilitate these functions when there is increased cell-cell and cell-matrix contact. GPR126 activity also increased after stachel treatment providing evidence for a positive feedback loop. This may also explain increased GPR126 expression in lung tissue from COPD patients. We did not directly test the relationship between GPR126 expression and cAMP signaling.
Differentially regulated genes after stachel activation include IL33, CTGF, and SERPINE1, all of which have known roles in airway disease and present possible mechanisms for GPR126 involvement in airway disease. Chronic inflammation of the airways is a key feature of many diseases causing airflow obstruction and airway smooth muscle secretes a variety of chemokines and cytokines that can perpetuate airway inflammation. 55 For example, IL33 (upregulated by GPR126 activation) is a well characterized cytokine that plays an important role in several chronic inflammatory diseases by promoting inflammatory cytokine production. 56 Serum levels of IL-33 and its receptor ST2 are elevated in patients with COPD compared to control subjects and peripheral blood lymphocytes from COPD patients release more IL33 in response to stimulus than healthy controls. 52 As GPR126 activation upregulated IL33 expression, reduced GPR126 expression in individuals carrying the protective alleles could potentially result in less IL33 being produced although this requires further investigation.
Airway remodeling results from a wide range of mechanisms involving smooth muscle cell hyperplasia, increased activation of fibroblasts and myofibroblasts, increased deposition of the ECM, and aberrant inflammatory responses. Airway smooth muscle undergoes alterations in health and disease characterized by cell proliferation, hypertrophy, and migration, and the expression of chemokines and cytokines is important in regulating airway inflammation. 55 Furthermore, smooth muscle mass is altered in COPD due to hypertrophy and hyperplasia. 55 We observed increased GPR126 expression in more confluent smooth muscle cells and, therefore, perhaps increased smooth muscle mass or ECM deposition in COPD is responsible for increased GPR126 expression observed in lung tissue from COPD patients.
CTGF is a gene downregulated by GPR126 activation which has roles in inflammation and tissue remodeling. This ECM protein recruits fibroblasts to wound sites, along with other inflammatory mediators such as TGF-β and IL-8/CXCL8. CTGF has a known role in pulmonary fibrosis and airway remodeling and has been shown to modulate a number of signaling pathways involved in cell adhesion and migration, angiogenesis, myofibroblast activation, and extracellular matrix deposition, which together can result in tissue remodeling and fibrosis. 51 These processes are also in keeping with what is known about adhesion GPCR biology. 10 Elevated CTGF expression contributes to expression of ECM proteins, cell migration, and the myofibroblastic phenotype in tissue repair. 57 In mouse lung tissue, CTGF expression and promoter activity are elevated after bleomycin challenge. 58 CTGF exerts profibrotic effects and contributes to lung fibrosis via transcriptional regulation of COL1A2. 58 Altered CTGF expression might seem unlikely to mediate the effects of GPR126 variants as expression was downregulated following GPR126 activation, although increased remodeling would be expected to lead to a reduced FVC and hence downregulation of CTGF could be working through this route.
SERPINE1 (PAI-1) (also downregulated by GPR126 activation) has been shown to be upregulated in emphysema lung and increased expression of PAI-1 suppresses fibrinolysis leading to pathological fibrin deposition and tissue damage. 50,59 Moreover, PAI-1 knockout mice display an emphysema-like phenotype, possibly due to an imbalance in protease-antiproteases activity in the lung--a known factor in emphysema development. 60 Elevated levels of PAI-1 are observed in the sputum of COPD patients and these levels were more elevated in more severe COPD cases. 61,62 PAI-1 plays a critical role in fibrosis development in a number of tissues including in the lung 63 and is produced by smooth muscle cells. 59 As with CTGF, the direction of effect following GPR126 activation initially seems inconsistent with altered SERPINE1 levels mediating effects of GPR126 activation but again this could be working through increased FVC rather than altered FEV 1 .
In addition to potential roles in inflammation and remodeling, GPR126 activation resulted in the differential regulation of genes within pathways involved in proliferation and cell cycle regulation. It has also been recently shown that GPR126 knockdown results in an increased proliferation of pulmonary artery smooth muscle cells where increased expression of GPR126 was associated with decreased proliferation. 64 These results are consistent with our suggestion that GPR126 plays a role in control of cell proliferation in the lungs.
There are limitations to our study. First, we were unable to detect GPR126 protein expression due to technical challenges and, therefore, we were unable to confirm cell surface expression of GPR126 in our recombinant cell system and in the primary cells. Moreover, as we were unable to detect signaling using identified GPR126 ligands we chose to use the GPR126 stachel peptide, which has been shown to cross-activate other receptors within and across aGPCR families. For example, peptides derived from GPR64 and GPR126 do cross-activate their respective receptors. 65 However, the peptide from GPR126 shows higher potency and efficacy on its originating receptor. To address this, we assessed GPR64 expression levels in HASM cells and HBECs prior to experimentation. Expression of GPR64 was at the lowest level of detection with FPKM <1 (Table  1). These data provided some confidence the signal was GPR126 derived, however we cannot completely exclude the possibility that GPR64 may make a minor contribution to signaling.
Collectively, these differentially expressed genes and pathways highlight a potential novel role for GPR126 in regulating the connected processes of airway remodeling and airway inflammation--both key processes in the pathophysiology of COPD. The genetic analyses suggest that GPR126 increased expression/activity may be damaging and we hypothesize that this could result in dysregulation of pathways involved in smooth muscle cell proliferation which may, therefore, contribute to airway disease.

ACKNOWLEDGMENTS
IS received the following grants; Hermes Fellowship (University of Nottingham, 2016-2017), British Lung Foundation (Grant PPRG15-5). IPH holds a NIHR Senior Investigator award. This work was partially supported by an MRC programme grant (G1000861). Lung tissue samples were obtained and research conducted under the approval of the Nottingham Health Science Biobank and Papworth Hospital Research Tissue Bank.