Caroline Swan

Identification of novel polymorphisms within the promoter region of the human β2 adrenergic receptor gene

By screening the 1470 bp 5′ to the start codon of the human β2 adrenergic receptor gene, we have identified a total of eight polymorphisms (−20 T→C, −47 T→C, −367 T→C, −468 C→G, −654 G→A, −1023 G→A, −1343 A→G and −1429 T→A c.f. β2 adrenergic receptor start codon). Transient transfection of 5′ flanking deletion luciferase reporter constructs demonstrated the majority of activity of the human β2 adrenergic gene 5′ flanking region to be present within a 549 bp fragment immediately upstream from the start codon. Because of linkage disequilibrium, some combinations of polymorphisms were particularly frequent. We transiently transfected COS‐7 cells with luciferase constructs under the control of the 549 bp of 5′ flanking DNA containing the two most frequent extended haplotypes in this region. Luciferase activity was significantly reduced in cells transfected with the ‘mutant’ construct (−20C, −47C, −367C, −468G) c.f. the ‘wild‐type’ construct (−20T, −47T, −367T, −468C). These data suggest that polymorphisms have the potential to alter human β2 adrenergic receptor gene expression.

Identifying and testing candidate genetic polymorphisms in the irritable bowel syndrome (IBS): association with TNFSF15 and TNFα

Objectives The postinfectious irritable bowel syndrome (PI-IBS) suggests that impaired resolution of inflammation could cause IBS symptoms. The authors hypothesised that polymorphisms in genes whose expression were altered by gastroenteritis might be linked to IBS with diarrhoea (IBS-D) which closely resembles PI-IBS. Design Part 1: 25 healthy volunteers (HVs), 21 patients 6 months after Campylobacter jejuni infection, 37 IBS-D and 19 IBS with constipation (IBS-C) underwent rectal biopsy for gene expression analysis and peripheral blood mononuclear cell cytokine production assessment. Part 2: Polymorphisms in genes whose expression was altered in Part 1 were assessed in 179 HV, 179 IBS-D, 122 IBS-C and 41 PI-IBS. Results Part 1: Mucosal expression of seven genes was altered in IBS: CCL11, CCL13, Calpain 8 and TNFSF15 increased while NR1D1, GPR161 and GABRE decreased with similar patterns after infection with C jejuni. Part 2: The authors assessed 21 known single nucleotide polymorphisms (SNPs) in these seven genes and one SNP in each of the TNFα and IL-10 genes. Three out of five TNFSF15 SNPs (rs6478108, rs6478109 and rs7848647) showed reduced minor allele frequency (MAF) (0.28, 0.27 and 0.27) in subjects with IBS-D compared with HV (0.38, 0.36 and 0.37; p=0.007, 0.015 and 0.007, respectively) confirming others recent findings. The authors also replicated the previously reported association of the TNFα SNP rs1800629 with PI-IBS which showed an increase in the MAF at 0.30 versus 0.19 for HV (p=0.04). Conclusion IBS-D and PI-IBS patients are associated with TNFSF15 and TNFα genetic polymorphisms which also predispose to Crohns disease suggesting possible common underlying pathogenesis.

Impaired Uptake of Serotonin by Platelets From Patients With Irritable Bowel Syndrome Correlates With Duodenal Immune Activation

BACKGROUND & AIMS Patients with irritable bowel syndrome with diarrhea (IBS-D) have increased mucosal serotonin (5-hydroxytryptamine [5-HT]) availability, possibly because immune activation reduces activity of the 5-HT transporter (SERT). We investigated the relationship between mucosal and platelet SERT and immune activation of the duodenal mucosa in patients with IBS-D. METHODS We quantified mucosal intraepithelial lymphocytes (IELs), mast cells, and enterochromaffin cells in blood samples, measured levels of SERT messenger RNA (mRNA) in mucosal samples, and assessed platelet uptake of 5-HT and platelet membrane binding of (3)H-paroxetine in samples from 29 healthy volunteers (HVs), 20 patients with IBS-D, and 20 untreated patients with celiac disease. RESULTS Patients with IBS-D or celiac disease had increased numbers of IELs and mast cells compared with HVs (both P < .001). Levels of SERT mRNA were reduced in the mucosa of patients with IBS-D or celiac disease and were inversely correlated with numbers of IELs (r = -0.72, P < .0001). Uptake of 5-HT by platelets from patients with IBS-D or celiac disease was reduced (mean, 17.1 ± 3.5 and 28.3 ± 4.1 nmol·min(-1)·mg(-1), respectively) compared with HVs (50.8 ± 8.0 nmol·min(-1)·mg(-1), P < .01 and P = .05, respectively). Binding of paroxetine to membranes of platelets from patients with IBS-D (median [interquartile range], 226 [92-405] fmol/mg protein) was significantly greater than that from HVs (109 [69-175] fmol/mg protein) and correlated inversely with platelet uptake of 5-HT (r = -0.62, P = .03). Tryptase release from incubated biopsy samples was significantly increased in patients with IBS-D (2.2 [0.42-3.5] vs 0.50 [0.25-0.86] ng·mL(-1)·mg(-1) for HVs; P = .03). CONCLUSIONS Platelet SERT is reduced in IBS-D and associated with reduced levels of SERT mRNA and duodenal immune activation.

Modulation of human airway smooth muscle proliferation by type 3 phosphodiesterase inhibition.

Elevation in cell cAMP content can inhibit mitogenic signaling in cultured human airway smooth muscle (HASM) cells. We studied the effects of the type 3-selective phosphodiesterase inhibitor siguazodan, the type 4-selective phosphodiesterase inhibitor rolipram, and the nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) on proliferation of cultured HASM cells. At concentrations selective for the type 3 phosphodiesterase isoform, siguazodan inhibited both [3H]thymidine incorporation (IC50 2 microM) and the increase in cell number (10 microM; 64% reduction) induced by platelet-derived growth factor-BB (20 ng/ml). These effects were mimicked by IBMX. At concentrations selective for type 4 phosphodiesterase inhibition, rolipram was without effect. A 20-min exposure to siguazodan and rolipram did not increase whole cell cAMP levels. However, in HASM cells transfected with a cAMP-responsive luciferase reporter (p6CRE/Luc), increases in cAMP-driven luciferase expression were seen with siguazodan (3.9-fold) and IBMX (16.5-fold). These data suggest that inhibition of the type 3 phosphodiesterase isoform present in airway smooth muscle results in inhibition of mitogenic signaling, possibly through an increase in cAMP-driven gene expression.Elevation in cell cAMP content can inhibit mitogenic signaling in cultured human airway smooth muscle (HASM) cells. We studied the effects of the type 3-selective phosphodiesterase inhibitor siguazodan, the type 4-selective phosphodiesterase inhibitor rolipram, and the nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) on proliferation of cultured HASM cells. At concentrations selective for the type 3 phosphodiesterase isoform, siguazodan inhibited both [3H]thymidine incorporation (IC50 2 μM) and the increase in cell number (10 μM; 64% reduction) induced by platelet-derived growth factor-BB (20 ng/ml). These effects were mimicked by IBMX. At concentrations selective for type 4 phosphodiesterase inhibition, rolipram was without effect. A 20-min exposure to siguazodan and rolipram did not increase whole cell cAMP levels. However, in HASM cells transfected with a cAMP-responsive luciferase reporter (p6CRE/Luc), increases in cAMP-driven luciferase expression were seen with siguazodan (3.9-fold) and IBMX (16.5-fold). These data suggest that inhibition of the type 3 phosphodiesterase isoform present in airway smooth muscle results in inhibition of mitogenic signaling, possibly through an increase in cAMP-driven gene expression.

Effects of a range of β2 adrenoceptor agonists on changes in intracellular cyclic AMP and on cyclic AMP driven gene expression in cultured human airway smooth muscle cells

The effects of the selective β2 adrenoceptor agonists salbutamol, terbutaline and salmeterol and the non‐selective β adrenoceptor agonist isoprenaline on [3H]‐cyclic AMP formation and cyclic AMP response element (CRE) driven luciferase expression, assessed using the construct p6CRE/luc, were studied in primary cultures of human airway smooth muscle (HASM) cells. Optimal transfection conditions for transient expression of pGL3 Control were 4 μg DNA/well71 in a 6 well plate and 1.8 μl Transfectam/μg DNA. Expression was maximal at 48–72 h. Salbutamol (maximum response 19%, EC50 0.6 μM), terbutaline (maximum response 38%, EC50 2.3 μM) and salmeterol (maximum response 18%, EC50 0.0012 μM) were all partial agonists for cyclic AMP formation compared with isoprenaline (EC50 0.08 μM). However, all of the β2 adrenoceptor agonists produced increases in CRE‐driven luciferase activity, in cultured HASM transfected with the vector p6CRE/luc, which were equivalent or greater (salmeterol) than those seen with isoprenaline. Both salbutamol and salmeterol were more potent at increasing luciferase expression than in elevating cyclic AMP levels in these cells. The potency ratios (EC50 (cyclic AMP)/EC50 (LUC)) for the agents studied were isoprenaline: 0.2 fold, terbutaline: 3 fold, salbutamol: 24 fold, salmeterol: 38 fold. These data suggest that important quantitative differences exist in the ability of β2 adrenoceptor agonists to increase whole cell cyclic AMP levels in airway smooth muscle and to drive gene expression via a CRE‐driven mechanism.

GSTCD and INTS12 regulation and expression in the human lung.

Genome-Wide Association Study (GWAS) meta-analyses have identified a strong association signal for lung function, which maps to a region on 4q24 containing two oppositely transcribed genes: glutathione S-transferase, C-terminal domain containing (GSTCD) and integrator complex subunit 12 (INTS12). Both genes were found to be expressed in a range of human airway cell types. The promoter regions and transcription start sites were determined in mRNA from human lung and a novel splice variant was identified for each gene. We obtained the following evidence for GSTCD and INTS12 co-regulation and expression: (i) correlated mRNA expression was observed both via Q-PCR and in a lung expression quantitative trait loci (eQTL) study, (ii) induction of both GSTCD and INTS12 mRNA expression in human airway smooth muscle cells was seen in response to TGFβ1, (iii) a lung eQTL study revealed that both GSTCD and INTS12 mRNA levels positively correlate with percent predicted FEV1, and (iv) FEV1 GWAS associated SNPs in 4q24 were found to act as an eQTL for INTS12 in a number of tissues. In fixed sections of human lung tissue, GSTCD protein expression was ubiquitous, whereas INTS12 expression was predominantly in epithelial cells and pneumocytes. During human fetal lung development, GSTCD protein expression was observed to be highest at the earlier pseudoglandular stage (10-12 weeks) compared with the later canalicular stage (17-19 weeks), whereas INTS12 expression levels did not alter throughout these stages. Knowledge of the transcriptional and translational regulation and expression of GSTCD and INTS12 provides important insights into the potential role of these genes in determining lung function. Future work is warranted to fully define the functions of INTS12 and GSTCD.

Functional Polymorphism and Differential Regulation of CYSLTR1 Transcription in Human Airway Smooth Muscle and Monocytes

Cysteinyl leukotrienes play an important role in the pathophysiology of many inflammatory disorders, including asthma. The aim of this study was to characterize the mechanisms underlying transcriptional regulation of the human cysteinyl leukotriene receptor 1 (hCYSLTR1) gene. 5′RACE was performed on human airway smooth muscle (HASM) and peripheral blood mononuclear cells. A 1128-bp region of the hCYSLTR1 main putative promoter was screened for polymorphisms by sequencing of 48 individuals. Luciferase reporter gene assays were performed using fragments of the core promoter (232 bp to 1128 bp) in HASM and THP1 cells. Three hCYSLTR1 transcripts were found, one representing 90% of all messenger RNA identified. The genomic location of the transcription start sites suggested there are two putative hCYSLTR1 promoters. The majority of the transcriptional activity of the main putative promoter was detected between −232 and −679 bp. Four single-nucleotide polymorphisms in strong linkage disequilibrium were found in the region studied: −561 (rs7066737), −642 (rs2806489), −781 (rs2637204), and −940 (rs321029), with three haplotypes observed. In THP1 cells, the G allele (−642) caused a twofold decrease in luciferase expression compared to the A allele. These data suggest that the majority of hCYSLTR1 transcripts in HASM and monocytes arise from a single promoter located immediately upstream of the 5′ untranslated region, although rarer transcripts can also occur. This study also raises the possibility that cell-type-dependent differences in transcriptional activity caused by the presence of specific haplotypes within the main CYSLTR1 promoter may be a predictor of disease risk or treatment response.

The Ser82 RAGE variant affects lung function and serum RAGE in smokers and sRAGE production in vitro

Introduction Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model. Methods Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified. Results Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production. Conclusions This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism.

Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP‐activated whole‐cell chloride currents in human bronchial epithelial cells

Chloride channels are known to play critical physiological roles in many cell types. Here, we describe the expression of anion channels using RNA Seq in primary cultures of human bronchial epithelial cells (hBECs). Chloride intracellular channel (CLIC) family members were the most abundant chloride channel transcripts, and CLIC1 showed the highest level of expression. In addition, we characterize the chloride currents in hBECs and determine how inhibition of CLIC1 via pharmacological and molecular approaches impacts these. We demonstrate that CLIC1 is able to modulate cyclic AMP‐induced chloride currents and suggest that CLIC1 modulation could be important for chloride homeostasis in this cell type.