10 research outputs found
Novel kinin B₁ receptor splice variant and 5'UTR regulatory elements are responsible for cell specific B₁ receptor expression.
The kinin B₁ receptor (B₁R) is rapidly upregulated after tissue trauma or inflammation and is involved in cancer and inflammatory diseases such as asthma. However, the role of the: promoter; a postulated alternative promoter; and spliced variants in airway epithelial and other lung cells are poorly understood. We identified, in various lung cell lines and leucocytes, a novel, naturally occurring splice variant (SV) of human B₁R gene with a shorter 5'untranslated region. This novel SV is ≈35% less stable than the wild-type (WT) transcript in lung adenocarcinoma cells (H2126), but does not influence translation efficiency. Cell-specific differences in splice variant expression were observed post des[Arg10]-kallidin stimulation with delayed upregulation of SV compared to WT suggesting potentially different regulatory responses to inflammation. Although an alternative promoter was not identified in our cell-lines, several cell-specific regulatory elements within the postulated alternative promoter region (negative response element (NRE) -1020 to -766 bp in H2126; positive response element (PRE) -766 to -410 bp in 16HBE; -410 to +1 region acts as a PRE in H2126 and NRE in 16HBE cells) were found. These findings reveal complex regulation of B₁R receptor expression in pulmonary cells which may allow future therapeutic manipulation in chronic pulmonary inflammation and cancer
B<sub>1</sub>R WT and SV expression following DAKD stimulation.
<p>Quantitative real-time PCR measurements of time (0, 3, 6 and 24 hr) and dose effect of DAKD (100 nM and 1000 nM) on B<sub>1</sub>R WT (A) and SV (B) expression in H2126 and on B<sub>1</sub>R WT expression in 16HBE (C). B<sub>1</sub>R mRNA level at 0 hr was set to 1. Data from 4 experiments performed in duplicates with mean ± SEM represented. DAKD treated samples were compared with non-treated, media only (NT) for each time point. Data was analysed using unpaired Student's t-test where *p<0.05 is considered statistically significant. **p<0.001</p
B<sub>1</sub>R transcript is differentially expressed across a range of cell lines.
<p>B<sub>1</sub>R mRNA expression was normalised to housekeeping gene SOD1 in human pulmonary cell lines as quantified by real time PCR. Data represents mean ± SEM from 3 independent experiments, each performed in duplicate.</p
Deletion B<sub>1</sub>R promoter constructs in HIGH expressing H2126 and LOW expressing 16HBE reveal regulatory regions.
<p>Summary of results from deletion constructs of B<sub>1</sub>R regulatory regions (A). The size of each regulatory region is indicated relative to start of exon III of B<sub>1</sub>R (+1). PRE = positive regulatory element, NRE = negative regulatory element. Relative luciferase activity of promoter deletion constructs transfected into human lung adenocarcinoma H2126 (B) and human bronchial epithelium 16HBE (C). Data presented as mean with error bars representing SEM. Data was analysed using one-way ANOVA and Tukey's post-hoc test on 4 independent experiments, each performed using at least triplicates. *p≤0.05 was considered statistically significant. Activity of CP-E2I2-Luc construct containing the B<sub>1</sub>R 5′ promoter and −1020 bp to +1 was set to 1.</p
B<sub>1</sub>R WT is more stable than B<sub>1</sub>R SV under basal conditions.
<p>Actinomycin D (Act D) mRNA decay of B<sub>1</sub>R WT and SV transcripts in H2126 measured at 0, 1, 3, 5h using real time PCR (Act D treatment at concentration of 5 µg/mL). Data plotted is mean±SEM from four independent experiments each performed at least in duplicates. Half-life of mRNA can be roughly estimated by determining the time required to reach 50% transcript level (shown by dotted lines). For more accurate assessment, the trendline equations obtained by plotting the graph are used to determine the half-life. In this graph, the equation for B<sub>1</sub>R WT is y = 100e<sup>−0.213x</sup> while for B<sub>1</sub>R SV is y = 100e<sup>−0.344x</sup>, where y is set to 50 (indicating 50% of transcript remaining) which will allow the calculation of x (indicating time required to reach 50% transcript level). From these equations, the half-life of B<sub>1</sub>R WT is 3.28 hr and 2.02 hr for B<sub>1</sub>R SV.</p
B<sub>1</sub>R SV does not affect translation efficiency.
<p>Translation efficiency of B<sub>1</sub>R WT and SV 5′ UTR measured using luciferase expression normalised to Renilla expression over time. Transfection of WT-luciferase and SV-luciferase constructs into normal lung bronchial epithelial, 16HBE (A), and lung adenocarcinoma, H2126 (B). Results are the average of five experiments with error bars representing SEM. There was no significant difference between the translation efficiency of B<sub>1</sub>R WT and B<sub>1</sub>R SV.</p
5′RACE PCR analysis of H2126 cDNA reveals multiple products.
<p>H2126 cDNA was amplified using the GeneRacer 5′nested primer and RT Rev 2 primer (A). Expected product size was 450 bp although at least 5 other bands were observed. Lanes 1 and 2: H2126 cDNA, Lanes 3 and 4: no template control. <b>Major transcription start sites (TSS) identified</b> in this study are labelled relative to translation start site (ATG) of NCBI published sequences of B<sub>1</sub>R (B). TSS of transcript D identified in this study is located 12 bp upstream of TSS on NCBI (NM_000710) but matches TSS identified by Yang & Polgar (1996)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087175#pone.0087175-Yang3" target="_blank">[23]</a>. In addition to the full-length wild type B<sub>1</sub>R transcript, a splice variant of B<sub>1</sub>R (transcript D and E) was also identified in this study. The TSS of this splice variant was at two primary locations; 12 bp and 4 bp upstream of NCBI sequence. Schematic presentation of identified wild type (WT) and splice variant (SV) transcripts and position of primers used in RT-qPCR to specifically amplify WT (B1R WT F) and SV (B1R SV F) (C). Forward primers are spanning the splice sites while common reverse primer (B1R Rev qPCR) located in exon 3 was used for amplification of both transcripts.</p
B<sub>1</sub>R splice variant (B<sub>1</sub>R SV) transcript and heteroduplex band is present in several cell types.
<p>Amplification of B<sub>1</sub>R WT and SV using primer set RT Fow and RT Rev (A) and RT Fow2A and RT Rev 1 (B). Amplification of loading control housekeeping gene superoxide dismutase 1 (SOD1) (C). Amplification of B<sub>1</sub>R WT and SV from human leucocytes using primers RT Fow and RT Rev (D) and amplification of SOD1 (E). A representative image of additional heteroduplex band located between the WT and SV band in HFLF. Three extra PCR cycles were used to match conditions used in G, but without addition of 10X fresh PCR mix (F). Heteroduplex band removed after addition of 10X fresh PCR mix followed by 3 PCR cycles (G). Lane 1: 100 bp ladder. PCR no template control (NTC). Human leucocytes (Leuc).</p