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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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Schematic representation of mechanisms involved in the induction of hyperresponsiveness in Human ASM. Cytokines and salmeterol up-regulate mRNA expression of the relevant GPCR leading to increased IP3 production in response to specific contractile agents (as demonstrated in the current manuscript). In addition, cytokines can up-regulate CD38 leading to subsequent increased cADPR production [9]. Second messengers IP3 and cADPR activate the Ryanodine and IP3 receptor respectively leading to elevated calcium release from intracellular stores and downstream contractile responses.
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Figure 8: Schematic representation of mechanisms involved in the induction of hyperresponsiveness in Human ASM. Cytokines and salmeterol up-regulate mRNA expression of the relevant GPCR leading to increased IP3 production in response to specific contractile agents (as demonstrated in the current manuscript). In addition, cytokines can up-regulate CD38 leading to subsequent increased cADPR production [9]. Second messengers IP3 and cADPR activate the Ryanodine and IP3 receptor respectively leading to elevated calcium release from intracellular stores and downstream contractile responses.

Mentions: In agreement with the proposed mechanism described in the current study, IFNγ has been shown to induce cysteinyl leukotriene receptor 1 (CysLTR1) mRNA expression in Human ASM with subsequent augmentation of LTD4 mediated Ca2+ signalling responses [26]. These data suggest that regulation of Human ASM responses to multiple contractile agents involves regulation at the GPCR locus. In the current study we did not identify any effect of dexamethasone or IL1β on Human ASM IPx signalling in contrast to others [15,38], which may reflect cell donor/experimental differences. Previous data has demonstrated that e.g. IL-13 can augment KCl induced force generation in mouse tracheal rings suggesting a regulatory mechanism downstream of that examined in the current study [13]. A mechanism to explain these observations has been identified involving cytokine activation leading to elevated CD38 and subsequent cyclic ADP ribose (cADPr) which activates the ryanodine receptor (RyR) and modulates the calcium response in Human ASM [9]. Therefore, it is likely that regulation at the GPCR locus demonstrated in the current study provides some specificity and amplification of Ca2+ responses via IP3 generation and that induction of CD38 also contributes to the overall magnitude of Ca2+ and contractile responses (see Figure 8). Our finding that IL-13 did not influence bradykinin induced IPx production or BDKRB2 mRNA levels may suggest that augmentation of this specific agonist response is more dependent on the CD38/cADPr pathway although we can not exclude the influence of different cell donors/experimental designs.


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)

Schematic representation of mechanisms involved in the induction of hyperresponsiveness in Human ASM. Cytokines and salmeterol up-regulate mRNA expression of the relevant GPCR leading to increased IP3 production in response to specific contractile agents (as demonstrated in the current manuscript). In addition, cytokines can up-regulate CD38 leading to subsequent increased cADPR production [9]. Second messengers IP3 and cADPR activate the Ryanodine and IP3 receptor respectively leading to elevated calcium release from intracellular stores and downstream contractile responses.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Schematic representation of mechanisms involved in the induction of hyperresponsiveness in Human ASM. Cytokines and salmeterol up-regulate mRNA expression of the relevant GPCR leading to increased IP3 production in response to specific contractile agents (as demonstrated in the current manuscript). In addition, cytokines can up-regulate CD38 leading to subsequent increased cADPR production [9]. Second messengers IP3 and cADPR activate the Ryanodine and IP3 receptor respectively leading to elevated calcium release from intracellular stores and downstream contractile responses.
Mentions: In agreement with the proposed mechanism described in the current study, IFNγ has been shown to induce cysteinyl leukotriene receptor 1 (CysLTR1) mRNA expression in Human ASM with subsequent augmentation of LTD4 mediated Ca2+ signalling responses [26]. These data suggest that regulation of Human ASM responses to multiple contractile agents involves regulation at the GPCR locus. In the current study we did not identify any effect of dexamethasone or IL1β on Human ASM IPx signalling in contrast to others [15,38], which may reflect cell donor/experimental differences. Previous data has demonstrated that e.g. IL-13 can augment KCl induced force generation in mouse tracheal rings suggesting a regulatory mechanism downstream of that examined in the current study [13]. A mechanism to explain these observations has been identified involving cytokine activation leading to elevated CD38 and subsequent cyclic ADP ribose (cADPr) which activates the ryanodine receptor (RyR) and modulates the calcium response in Human ASM [9]. Therefore, it is likely that regulation at the GPCR locus demonstrated in the current study provides some specificity and amplification of Ca2+ responses via IP3 generation and that induction of CD38 also contributes to the overall magnitude of Ca2+ and contractile responses (see Figure 8). Our finding that IL-13 did not influence bradykinin induced IPx production or BDKRB2 mRNA levels may suggest that augmentation of this specific agonist response is more dependent on the CD38/cADPr pathway although we can not exclude the influence of different cell donors/experimental designs.

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