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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.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 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.

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 on H1 Histamine Receptor and B2 Bradykinin Receptor mRNA expression in Human ASM. 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).
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Figure 1: Effect of salmeterol or cytokine treatment on H1 Histamine Receptor and B2 Bradykinin Receptor mRNA expression in Human ASM. 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).

Mentions: In order to test the hypothesis that the site of regulation determining the alteration in IPx signalling observed following cytokine or drug pre-treatment occurs at the receptor locus we determined the mRNA expression levels of the H1 Histamine and B2 Bradykinin Receptors in our samples using Real Time PCR, i.e. the two key receptors mediating histamine or bradykinin activation of human ASM cells ([29,38], Figure 1). Quantification of the HRH1 mRNA levels identified a significant induction of gene expression following drug or cytokine treatment for 4 (p < 0.0001) or 24 hours (p < 0.0001) (Figures 1A and 1B). A significantly elevated level of HRH1 mRNA expression was observed following 4 hours treatment of Human ASM with IL-13 (254 +/- 14.5% compared to medium control (100%)) or IFNγ(228.7 +/- 30.9%). At 24 hours the IL-13 treated cells maintained an elevated level of HRH1 mRNA expression (298.5 +/- 17.0%) and salmeterol treated cells demonstrated a significantly elevated level of HRH1 mRNA expression (217.3 +/- 47.1%). Quantification of BDKRB2 mRNA expression in Human ASM cells following cytokine or drug treatment identified a significant effect on receptor mRNA expression at 4 (p < 0.0001) and 24 hours (p < 0.0001) post treatment (Figures 1C and 1D). A significant induction of BDKRB2 mRNA expression was observed at 4 hours following TNFα (461.0 +/- 20.4%) or IFNγ (322.1 +/- 53.3%) treatment. Following 24 hours treatment of ASM cells an elevated level of BDKRB2 mRNA was observed in the TNFα (156.2 +/- 6.5) and salmeterol (147.1 +/- 14.7%) treated cells. Treatment of Human ASM cells with IL-13 did not significantly influence BDKRB2 mRNA expression.


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 on H1 Histamine Receptor and B2 Bradykinin Receptor mRNA expression in Human ASM. 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Effect of salmeterol or cytokine treatment on H1 Histamine Receptor and B2 Bradykinin Receptor mRNA expression in Human ASM. 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).
Mentions: In order to test the hypothesis that the site of regulation determining the alteration in IPx signalling observed following cytokine or drug pre-treatment occurs at the receptor locus we determined the mRNA expression levels of the H1 Histamine and B2 Bradykinin Receptors in our samples using Real Time PCR, i.e. the two key receptors mediating histamine or bradykinin activation of human ASM cells ([29,38], Figure 1). Quantification of the HRH1 mRNA levels identified a significant induction of gene expression following drug or cytokine treatment for 4 (p < 0.0001) or 24 hours (p < 0.0001) (Figures 1A and 1B). A significantly elevated level of HRH1 mRNA expression was observed following 4 hours treatment of Human ASM with IL-13 (254 +/- 14.5% compared to medium control (100%)) or IFNγ(228.7 +/- 30.9%). At 24 hours the IL-13 treated cells maintained an elevated level of HRH1 mRNA expression (298.5 +/- 17.0%) and salmeterol treated cells demonstrated a significantly elevated level of HRH1 mRNA expression (217.3 +/- 47.1%). Quantification of BDKRB2 mRNA expression in Human ASM cells following cytokine or drug treatment identified a significant effect on receptor mRNA expression at 4 (p < 0.0001) and 24 hours (p < 0.0001) post treatment (Figures 1C and 1D). A significant induction of BDKRB2 mRNA expression was observed at 4 hours following TNFα (461.0 +/- 20.4%) or IFNγ (322.1 +/- 53.3%) treatment. Following 24 hours treatment of ASM cells an elevated level of BDKRB2 mRNA was observed in the TNFα (156.2 +/- 6.5) and salmeterol (147.1 +/- 14.7%) treated cells. Treatment of Human ASM cells with IL-13 did not significantly influence BDKRB2 mRNA expression.

Bottom Line: 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 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.

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