Effect of salmeterol or cytokine treatment on H1 Histamine Receptor and B2 Bradykinin Receptor mRNA expression in Human ASM

<p><b>Copyright information:</b></p><p>Taken from "Salmeterol and cytokines modulate inositol-phosphate signalling in Human airway smooth muscle cells via regulation at the receptor locus"</p><p>Respiratory Research 2007;8(1):68-68.</p><p>Published online 28 Sep 2007</p><p>PMCID:PMC2117012.</p><p></p> ASM cells were serum starved for 24 hours and then treated with medium alone, 10 ng/ml TNFα, 10 ng/ml IFNγ, 50 ng/ml IL-13 or 1 μM salmeterol for 4 or 24 hours. mRNA levels of the H1 Histamine Receptor and B2 Bradykinin Receptor were quantified using Real Time PCR and normalised using the 18s ribosomal RNA endogenous control. HRH1 mRNA quantification following 4 hours (A) and 24 hours (B) stimulation. BDKRB2 mRNA quantification following 4 hours (C) and 24 hours (D) stimulation. Data is normalised to medium control = 100%, n = 3 independent experiments in triplicate, mean +/- S.E.M. Dunnett's Multiple Comparison Test (*p < 0.05)

Effect of salmeterol or cytokine treatment for 4 hours on promoter activity in Human ASM cells transfected with -Luciferase constructs

<p><b>Copyright information:</b></p><p>Taken from "Salmeterol and cytokines modulate inositol-phosphate signalling in Human airway smooth muscle cells via regulation at the receptor locus"</p><p>Respiratory Research 2007;8(1):68-68.</p><p>Published online 28 Sep 2007</p><p>PMCID:PMC2117012.</p><p></p> Human ASM cells were transfected and stimulated as described in Figure 4 except derivatives of pGL4-Luc2 containing the BDKRB2 inserts were used. Following 4 hours treatment cells were harvested and firefly luciferase was quantified. pGL4-BDKRB2-1kb-Luc2 (A), pGL4-BDKRB2-2kb-Luc2 (B), pGL4-BDKRB2-3kb-Luc2 (C), pGL4-BDKRB2-4kb-Luc2 (D) transfections. Data is normalised to the mean luciferase activity of each construct transfection treated with medium alone +/- S.E.M. (n = 4 independent experiments). Dunnett's Multiple Copmarison Test (**p < 0.01)

Effect of salmeterol or cytokine treatment for 4 hours on promoter activity in Human ASM cells transfected with -Luciferase or control constructs

<p><b>Copyright information:</b></p><p>Taken from "Salmeterol and cytokines modulate inositol-phosphate signalling in Human airway smooth muscle cells via regulation at the receptor locus"</p><p>Respiratory Research 2007;8(1):68-68.</p><p>Published online 28 Sep 2007</p><p>PMCID:PMC2117012.</p><p></p> Complete medium was replaced with serum free medium and cells were transfected with 0.12 μg of pGL4-Luc2 using Fugene 6 (Roche) at a 3:1 ratio (Fugene:DNA). For derivatives of pGL4-Luc2 containing the HRH1 or SV40 control inserts equimolar amounts of DNA were used. Cells were allowed to grow for 16 hours prior to the addition of medium alone, 10 ng/ml TNFα, 10 ng/ml IFNγ, 50 ng/ml IL-13 or 1 μM salmeterol. Following 4 hours treatment cells were harvested and firefly luciferase was quantified. pGL4-Luc2 (A), pGL4-SV40-Luc2 (B), pGL4-HRH1-1 kb-Luc2 (C), pGL4-HRH1-2 kb-Luc2 (D), pGL4-HRH1-3 kb-Luc2 (E), pGL4-HRH1-4 kb-Luc2 (F) transfections. Data is normalised to the mean luciferase activity of each construct transfection treated with medium alone +/- S.E.M. (n = 4 independent experiments). Dunnett's Multiple Comparison Test (*p < 0.05)

A: Expression of <i>GSTCD</i> and <i>INTS12</i> mRNA in Lung and Airway cells.

<p>mRNA expression in human airway smooth muscle (HASM) cells, human bronchial epithelial cells (HBEC) and peripheral blood mononuclear cells (PBMC) is shown relative to mRNA from lung. Open bars depict <i>GSTCD</i> expression whereas black bars show <i>INTS12</i> expression. Values shown are mean and standard error of the mean (SEM) (n=3). Only the expression of <i>GSTCD</i> in HBEC relative to lung was statistically significant (* <i>P</i>=0.0494). <b>B</b>: <b>Correlation between <i>GSTCD</i> and <i>INTS12</i> ΔCt values in HASM, HBEC, PBMC and lung</b>. mRNA expression levels as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#pone-0074630-g003" target="_blank">Figure 3A</a> from human airway smooth muscle (HASM) cells, human bronchial epithelial cells (HBEC), peripheral blood mononuclear cells (PBMC) and lung were correlated using a scatter plot. The correlation coefficient between these measures was r=0.8, <i>P</i><0.0001. <b>C</b>: <b>Correlation between <i>GSTCD</i> and <i>INTS12</i> mRNA levels in the lung</b>. The scatter plot shows a positive correlation between the <i>GSTCD</i> and <i>INTS12</i> probe sets as investigated in the lung eQTL study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#B20" target="_blank">20</a>].</p

<i>GSTCD</i> and <i>INTS12</i> gene expression is altered following exposure of HASM cells to TGFβ1.

<p>Human airway smooth muscle (HASM) cells were exposed to 10ng/ml TGFβ1 for 4 or 24 hours. Open bars depict <i>GSTCD</i> expression whereas black bars show <i>INTS12</i> expression. Values shown are mean and standard error of the mean (SEM) (n=5). Significant increases in both <i>GSTCD</i> and <i>INTS12</i> gene expression were observed following 24h exposure to TGFβ1 (<i>P</i><0.05 <i>GSTCD</i>, <i>P</i><0.01 <i>INTS12</i>) and after 4h TGFβ1 exposure in <i>INTS12</i> expression (<i>P</i><0.05).</p

Correlation of <i>GSTCD</i> (A) and <i>INTS12</i> (B) lung mRNA levels with percent predicted FEV₁ in the lungs of 848 individuals (see Table S5 for patient demographics).

<p>Correlation of <i>GSTCD</i> (A) and <i>INTS12</i> (B) lung mRNA levels with percent predicted FEV₁ in the lungs of 848 individuals (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#pone.0074630.s009" target="_blank">Table S5</a> for patient demographics).</p

Regulatory motifs within the <i>GSTCD/INTS12</i> locus.

<p>The <i>GSTCD</i>/<i>INTS12</i> locus is shown, annotated with RNA sequencing, H3K27Ac histone marks, DNase hypersensitivity, transcription factor binding and CpG islands (UCSC Genome Browser (<a href="http://genome.ucsc.edu/" target="_blank"><u>http://genome.ucsc.edu/</u></a>)) on the Human Feb 2009 (GRCh37/hg19) assembly. For the H3K27Ac histone marks and RNA sequence tracks, peak height is proportional to signal amplitude, with colours representing datasets in different cell backgrounds (pale blue H3K27Ac histone trace = human umbilical vein endothelial cell (HUVEC); blue/grey = K562 erythroleukaemia cells). For the DNase hypersensitivity and transcription factor binding tracks, a grey band indicates the extent of the hypersensitive region and the intensity of the band is proportional to the maximum signal strength observed in any cell line.</p

Genetic architecture of the region containing both <i>GSTCD</i> and <i>INTS12</i> genes.

<p>The top panel depicts gene arrangements previously reported in NCBI, build 37, whereas the lower panel shows novel variants identified in lung. V1, 2, 3 refer to splice variants 1, 2 and 3 for each gene. Open boxes represent exons and connecting black lines represent introns. Also illustrated are the locations of Single Nucleotide Polymorphisms (SNPs) meeting genome-wide association (<i>P</i>≤5x10^-8) for FEV₁ in previously reported analyses of the SpiroMeta consortium [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#B5" target="_blank">5</a>]. Highlighted in red is the sentinel SNP rs10516526 that was associated with FEV₁ (<i>P</i>=2.18 x 10^-23 in all stage analyses) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074630#B5" target="_blank">5</a>]. Translation start codons (ATG) are shown boxed in green and stop codons (TAA) boxed in red.</p

GSTCD and INTS12 protein expression in human tissue.

<p>Immunohistochemistry studies assessed GSTCD and INTS12 protein expression in tissue sections from controls, individuals with COPD and from a range of fetal developmental stages. All images x40 magnification. <b>A</b>: Representative images of GSTCD expression in lung tissue from three healthy donors (images a and e) with matched isotype controls (iso, images c and g) and in lung tissue from three donors with COPD (images b and f) with matched isotype controls (iso, images d and h). <b>B</b>: Representative images of INTS12 expression in lung tissue from three control donors (images a and e) with matched isotype controls (iso, images c and g) and in lung tissue from three donors with COPD (images b and f) with matched isotype controls (iso, images d and h). <b>C</b>: Panel 1: GSTCD expression in tissue from human fetuses at a range of developmental stages: embryonic (19 days, a and b; 21 days, c and d; 23 days, e and f); pseudoglandular (10 weeks, g and h; 12 weeks, i and j); canalicular (17 weeks, k; 19 weeks, l). Expression was observed to be increased through the pseudoglandular stage. Panel 2: INTS12 expression in the same panel of tissue samples as described above. Isotype controls were all negative (data not shown).</p

<i>AGER</i> isoform expression in three HBEC donors using RNA Seq.

<p>Structure and abundance of known <i>AGER</i> isoforms in three human bronchial epithelial cell donors illustrating heterogeneity in expression levels. Percentage abundances (% FPKM) were calculated for each donor. Transcripts for full length and soluble <i>AGER</i> were identified at similar low abundancies. FPKM; fragments per kilobase of transcript per million mapped reads.</p