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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 4 hours on BDKRB2 promoter activity in Human ASM cells transfected with BDKRB2-Luciferase constructs. 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).
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Figure 6: Effect of salmeterol or cytokine treatment for 4 hours on BDKRB2 promoter activity in Human ASM cells transfected with BDKRB2-Luciferase constructs. 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).

Mentions: In an analogous manner to that described for the HRH1 promoter analyses the effect of TNFα, IFNγ or salmeterol on BDKRB2 mediated transcription was evaluated using the pGL4-BDKRB2-luciferase transfected Human ASM cells. Following 4 hours stimulation with TNFα or IFNγ there were no apparent effects on BDKRB2 mediated transcription for the 1, 2, 3 and 4 kb constructs. Salmeterol treatment resulted in an augmentation of BDKRB2 mediated transcription for the 1, 2, 3 and 4 kb constructs (409.4 +/- 65.7, 446.7 +/- 111.9, 245.3 +/- 30.8 and 542.4 +/- 148.5 respectively, p < 0.01, Figure 6). Following 24 hours stimulation of BDKRB2-luciferase transfected Human ASM cells with TNFα, IFNγ or salmeterol a similar pattern to the 4 hour experiments was observed. TNFα and IFNγ stimulation did not significantly influence BDKRB2 mediated transcription (Figure 7). Salmeterol treatment of BDKRB2-luciferase transfected Human ASM cells for 24 hours significantly augmented BDKRB2 mediated transcription for the 1, 2 and 4 kb constructs (190.9 +/- 30.0, 208.8 +/- 42.7 and 377.7 +/- 110.7 respectively, p < 0.05, Figures 7A, 7B, 7D), however, the effect was apparent for all constructs.


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 4 hours on BDKRB2 promoter activity in Human ASM cells transfected with BDKRB2-Luciferase constructs. 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2117012&req=5

Figure 6: Effect of salmeterol or cytokine treatment for 4 hours on BDKRB2 promoter activity in Human ASM cells transfected with BDKRB2-Luciferase constructs. 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).
Mentions: In an analogous manner to that described for the HRH1 promoter analyses the effect of TNFα, IFNγ or salmeterol on BDKRB2 mediated transcription was evaluated using the pGL4-BDKRB2-luciferase transfected Human ASM cells. Following 4 hours stimulation with TNFα or IFNγ there were no apparent effects on BDKRB2 mediated transcription for the 1, 2, 3 and 4 kb constructs. Salmeterol treatment resulted in an augmentation of BDKRB2 mediated transcription for the 1, 2, 3 and 4 kb constructs (409.4 +/- 65.7, 446.7 +/- 111.9, 245.3 +/- 30.8 and 542.4 +/- 148.5 respectively, p < 0.01, Figure 6). Following 24 hours stimulation of BDKRB2-luciferase transfected Human ASM cells with TNFα, IFNγ or salmeterol a similar pattern to the 4 hour experiments was observed. TNFα and IFNγ stimulation did not significantly influence BDKRB2 mediated transcription (Figure 7). Salmeterol treatment of BDKRB2-luciferase transfected Human ASM cells for 24 hours significantly augmented BDKRB2 mediated transcription for the 1, 2 and 4 kb constructs (190.9 +/- 30.0, 208.8 +/- 42.7 and 377.7 +/- 110.7 respectively, p < 0.05, Figures 7A, 7B, 7D), however, the effect was apparent for all constructs.

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