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Salmeterol and cytokines modulate inositol-phosphate signalling in human airway smooth muscle cells via regulation at the receptor locus.

Smith N, Browning CA, Duroudier N, Stewart C, Peel S, Swan C, Hall IP, Sayers I - Respir. Res. (2007)

Bottom Line: No treatment significantly influenced sodium fluoride induced IPx responses suggesting regulation occurs at the receptor locus.The effect of cytokines on HRH1 and BDKRB2 promoter-reporter expression suggested a more complex regulation of mRNA expression involving additional loci to the core promoter.These data provide further insight into the molecular basis of AHR and extend our understanding of potentially detrimental effects associated with existing therapies used in the treatment of asthma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Therapeutics & Molecular Medicine, University Hospital of Nottingham, Nottingham, UK. mzywnas@nottingham.ac.uk

ABSTRACT

Background: Airway hyper-responsiveness (AHR) is a key feature of asthma and a causal relationship between airway inflammation and AHR has been identified. The aim of the current study was to clarify the effect of proinflammatory cytokines and asthma medication on primary human airway smooth muscle (ASM) inositol phosphate (IPx) signalling and define the regulatory loci involved.

Methods: Primary Human ASM cells were isolated from explants of trachealis muscle from individuals with no history of respiratory disease. The effect of cytokine or asthma medication on histamine or bradykinin induced IPx signalling was assessed by [3H] inositol incorporation. Quantitative Real Time PCR was used to measure mRNA levels of receptors and downstream signalling components. Transcriptional mechanisms were explored using a combination of 5'Rapid Amplification of cDNA Ends (5'RACE) and promoter-reporter techniques.

Results: Treatment of Human ASM cells with IL-13, IFN gamma or salmeterol for 24 hours lead to a modest augmentation of histamine induced IPx responses (144.3 +/- 9.3, 126.4 +/- 7.5 and 117.7 +/- 5.2%, p < 0.05). Similarly, TNFalpha, IFN gamma or salmeterol treatment augmented bradykinin induced IPx responses (127.4 +/- 8.3, 128.0 +/- 8.4 and 111.7 +/- 5.0%, P < 0.05). No treatment significantly influenced sodium fluoride induced IPx responses suggesting regulation occurs at the receptor locus. Analyses of mRNA expression of components of the IPx pathway i.e. H1 Histamine Receptor (HRH1), B2 Bradykinin Receptor (BDKRB2), G alpha q/11 and PLC-beta1 identified that a significant induction of receptor mRNA (>2 fold) was a feature of these responses explaining the cytokine and spasmogen specificity. The HRH1 and BDKRB2 promoter regions were mapped in ASM and promoter-reporter analyses identified that salmeterol can induce HRH1 (>2 fold) and BDKRB2 (2-5 fold) transcription. The effect of cytokines on HRH1 and BDKRB2 promoter-reporter expression suggested a more complex regulation of mRNA expression involving additional loci to the core promoter.

Conclusion: Our results indicate that the spasmogen specific receptor locus may be a key site of regulation determining the magnitude of spasmogen mediated ASM IPx responses during airway inflammation or following asthma medication. These data provide further insight into the molecular basis of AHR and extend our understanding of potentially detrimental effects associated with existing therapies used in the treatment of asthma.

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Effect of salmeterol or cytokine treatment for 24 hours on HRH1 promoter activity in Human ASM cells transfected with HRH1-Luciferase or control constructs. Human ASM cells were transfected and stimulated as described in Figure 4. Following 24 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 = 5 independent experiments) Dunnett's Multiple Copmarison Test (**p < 0.01).
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Figure 5: Effect of salmeterol or cytokine treatment for 24 hours on HRH1 promoter activity in Human ASM cells transfected with HRH1-Luciferase or control constructs. Human ASM cells were transfected and stimulated as described in Figure 4. Following 24 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 = 5 independent experiments) Dunnett's Multiple Copmarison Test (**p < 0.01).

Mentions: In order to investigate the effect of cytokines or salmeterol on HRH1 promoter activity in Human ASM, cells were transfected with the series of pGL4-HRH1 constructs for 16 hours and then stimulated with the appropriate cytokine or salmeterol for 4 (Figure 4) or 24 (Figure 5) hours prior to luciferase quantification. These analyses were not susceptible to transfection efficiency bias as comparisons were made within transfections. Predominantly, for these pGL4-Luc2 control transfections the background luciferase activity was not affected by stimulation with cytokines or salmeterol, however, a significant reduction in pGL4-Luc2 mediated luciferase activity was observed following 24 hours TNFα treatment (37.4 +/- 5.0 compared to medium control (100%), Figure 5A). Stimulation of Human ASM cells transfected with pGL4-SV40-Luc2 control plasmid did not identify any effect of TNFα, IFNγ, IL-13 or salmeterol treatment for 4 and 24 hours on SV40 mediated luciferase production (Figures 4B and 5B). Analyses of Human ASM cells transfected with the HRH1 1, 2, 3 and 4 kb promoter constructs and stimulated for 4 hours with IFNγ, IL-13 or salmeterol provided evidence that the level of transcription was influenced by treatment for the 3 and 4 kb constructs (ANOVA, p = 0.05 and p = 0.05 respectively Figures 4E and 4F). However, following post-hoc analyses only salmeterol significantly augmented transcription of the 4 kb core HRH1 promoter (224.5 +/- 37.4 versus medium control (100%), p < 0.05 Figure 4F). There was suggestive evidence that IL-13 may influence transcription of the HRH1 3 kb promoter (204.1 +/- 50.1) and IFNγ may influence the 4 kb construct (164.4 +/- 36.8, Figures 4E and 4F) although these differences were not statistically significant. Analyses of Human ASM cells transfected with the HRH1 1, 2, 3 and 4 kb promoter constructs and stimulated for 24 hours with IFNγ, IL-13 or salmeterol provided evidence that the level of transcription was affected for the 1 kb constructs (ANOVA, p = 0.03, Figure 5C) and that salmeterol significantly augments the HRH1 1 kb construct transcriptional activity (250.3 +/- 60.3, p < 0.05, Figure 5C). As previously, there was evidence that IL-13 influences transcription of the HRH1 3 kb promoter (205.3 +/- 90.2, Figure 5E).


Salmeterol and cytokines modulate inositol-phosphate signalling in human airway smooth muscle cells via regulation at the receptor locus.

Smith N, Browning CA, Duroudier N, Stewart C, Peel S, Swan C, Hall IP, Sayers I - Respir. Res. (2007)

Effect of salmeterol or cytokine treatment for 24 hours on HRH1 promoter activity in Human ASM cells transfected with HRH1-Luciferase or control constructs. Human ASM cells were transfected and stimulated as described in Figure 4. Following 24 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 = 5 independent experiments) Dunnett's Multiple Copmarison Test (**p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 5: Effect of salmeterol or cytokine treatment for 24 hours on HRH1 promoter activity in Human ASM cells transfected with HRH1-Luciferase or control constructs. Human ASM cells were transfected and stimulated as described in Figure 4. Following 24 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 = 5 independent experiments) Dunnett's Multiple Copmarison Test (**p < 0.01).
Mentions: In order to investigate the effect of cytokines or salmeterol on HRH1 promoter activity in Human ASM, cells were transfected with the series of pGL4-HRH1 constructs for 16 hours and then stimulated with the appropriate cytokine or salmeterol for 4 (Figure 4) or 24 (Figure 5) hours prior to luciferase quantification. These analyses were not susceptible to transfection efficiency bias as comparisons were made within transfections. Predominantly, for these pGL4-Luc2 control transfections the background luciferase activity was not affected by stimulation with cytokines or salmeterol, however, a significant reduction in pGL4-Luc2 mediated luciferase activity was observed following 24 hours TNFα treatment (37.4 +/- 5.0 compared to medium control (100%), Figure 5A). Stimulation of Human ASM cells transfected with pGL4-SV40-Luc2 control plasmid did not identify any effect of TNFα, IFNγ, IL-13 or salmeterol treatment for 4 and 24 hours on SV40 mediated luciferase production (Figures 4B and 5B). Analyses of Human ASM cells transfected with the HRH1 1, 2, 3 and 4 kb promoter constructs and stimulated for 4 hours with IFNγ, IL-13 or salmeterol provided evidence that the level of transcription was influenced by treatment for the 3 and 4 kb constructs (ANOVA, p = 0.05 and p = 0.05 respectively Figures 4E and 4F). However, following post-hoc analyses only salmeterol significantly augmented transcription of the 4 kb core HRH1 promoter (224.5 +/- 37.4 versus medium control (100%), p < 0.05 Figure 4F). There was suggestive evidence that IL-13 may influence transcription of the HRH1 3 kb promoter (204.1 +/- 50.1) and IFNγ may influence the 4 kb construct (164.4 +/- 36.8, Figures 4E and 4F) although these differences were not statistically significant. Analyses of Human ASM cells transfected with the HRH1 1, 2, 3 and 4 kb promoter constructs and stimulated for 24 hours with IFNγ, IL-13 or salmeterol provided evidence that the level of transcription was affected for the 1 kb constructs (ANOVA, p = 0.03, Figure 5C) and that salmeterol significantly augments the HRH1 1 kb construct transcriptional activity (250.3 +/- 60.3, p < 0.05, Figure 5C). As previously, there was evidence that IL-13 influences transcription of the HRH1 3 kb promoter (205.3 +/- 90.2, Figure 5E).

Bottom Line: No treatment significantly influenced sodium fluoride induced IPx responses suggesting regulation occurs at the receptor locus.The effect of cytokines on HRH1 and BDKRB2 promoter-reporter expression suggested a more complex regulation of mRNA expression involving additional loci to the core promoter.These data provide further insight into the molecular basis of AHR and extend our understanding of potentially detrimental effects associated with existing therapies used in the treatment of asthma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Therapeutics & Molecular Medicine, University Hospital of Nottingham, Nottingham, UK. mzywnas@nottingham.ac.uk

ABSTRACT

Background: Airway hyper-responsiveness (AHR) is a key feature of asthma and a causal relationship between airway inflammation and AHR has been identified. The aim of the current study was to clarify the effect of proinflammatory cytokines and asthma medication on primary human airway smooth muscle (ASM) inositol phosphate (IPx) signalling and define the regulatory loci involved.

Methods: Primary Human ASM cells were isolated from explants of trachealis muscle from individuals with no history of respiratory disease. The effect of cytokine or asthma medication on histamine or bradykinin induced IPx signalling was assessed by [3H] inositol incorporation. Quantitative Real Time PCR was used to measure mRNA levels of receptors and downstream signalling components. Transcriptional mechanisms were explored using a combination of 5'Rapid Amplification of cDNA Ends (5'RACE) and promoter-reporter techniques.

Results: Treatment of Human ASM cells with IL-13, IFN gamma or salmeterol for 24 hours lead to a modest augmentation of histamine induced IPx responses (144.3 +/- 9.3, 126.4 +/- 7.5 and 117.7 +/- 5.2%, p < 0.05). Similarly, TNFalpha, IFN gamma or salmeterol treatment augmented bradykinin induced IPx responses (127.4 +/- 8.3, 128.0 +/- 8.4 and 111.7 +/- 5.0%, P < 0.05). No treatment significantly influenced sodium fluoride induced IPx responses suggesting regulation occurs at the receptor locus. Analyses of mRNA expression of components of the IPx pathway i.e. H1 Histamine Receptor (HRH1), B2 Bradykinin Receptor (BDKRB2), G alpha q/11 and PLC-beta1 identified that a significant induction of receptor mRNA (>2 fold) was a feature of these responses explaining the cytokine and spasmogen specificity. The HRH1 and BDKRB2 promoter regions were mapped in ASM and promoter-reporter analyses identified that salmeterol can induce HRH1 (>2 fold) and BDKRB2 (2-5 fold) transcription. The effect of cytokines on HRH1 and BDKRB2 promoter-reporter expression suggested a more complex regulation of mRNA expression involving additional loci to the core promoter.

Conclusion: Our results indicate that the spasmogen specific receptor locus may be a key site of regulation determining the magnitude of spasmogen mediated ASM IPx responses during airway inflammation or following asthma medication. These data provide further insight into the molecular basis of AHR and extend our understanding of potentially detrimental effects associated with existing therapies used in the treatment of asthma.

Show MeSH
Related in: MedlinePlus