Limits...
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.

Show MeSH

Related in: MedlinePlus

Transcriptional activity of HRH1 and BDKRB2 core promoter fragments. Luciferase activity in Human ASM cell lysates 20 hours post transfection with control, HRH1 and BDKRB2 luciferase constructs (A and B) (n = 4), Luciferase activity in Human ASM cell lysates 40 hours post transfection with control, HRH1 and BDKRB2 luciferase constructs (C and D) (n = 5). Dunnett's Multiple Comparison Test (compared to pGL4-Luc2 control **p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2117012&req=5

Figure 3: Transcriptional activity of HRH1 and BDKRB2 core promoter fragments. Luciferase activity in Human ASM cell lysates 20 hours post transfection with control, HRH1 and BDKRB2 luciferase constructs (A and B) (n = 4), Luciferase activity in Human ASM cell lysates 40 hours post transfection with control, HRH1 and BDKRB2 luciferase constructs (C and D) (n = 5). Dunnett's Multiple Comparison Test (compared to pGL4-Luc2 control **p < 0.01).

Mentions: Preliminary luciferase experiments using CHO-K1 cells transfected with pGL4 constructs for 40 hours provided clear evidence that all of the HRH1 and BDKRB2 promoter-luciferase plasmids were transcriptionally active (all >15 fold over pGL4-Luc2 control, p = 0.0002 and p = 0.0004 respectively, data not shown). Interestingly the greatest activity in CHO-K1 cells was observed for the plasmids containing the longest segment of the two core promoters i.e. the HRH1-4 kb (106.5 +/- 34.3 fold over pGL4-Luc2, p < 0.01) and BDKRB2-4 kb (219.4 +/- 75.3 fold over pGL4-Luc2, p < 0.01) plasmids. Basal activity of HRH1 promoter containing plasmids transfected into Human ASM showed low level activity over pGL4-Luc2 at 20 hours post transfection with maximal activity observed for the pGL4-HRH1-2 kb transfection samples (2.1 +/- 0.2 fold, Figure 3A). Analyses of luciferase activity in Human ASM cells transfected with the series of BDKRB2 or control constructs for 20 hours demonstrated significant differences in activity between constructs (ANOVA, p = 0.0001). The BDKRB2 1 kb (5.6 +/- 0.9 fold) and 4 kb (3.8 +/- 0.8 fold) constructs showed clear activity (Figure 3B). The pGL4-BDKRB2-3 kb transfection samples showed similar to pGL4-Luc2 activity (Figure 3B). Transfection of Human ASM cells with HRH1 promoter containing constructs for 40 hours again demonstrated a modest transcriptional activity over pGL4-Luc2 as observed for the 20 hour transfections (maximum HRH1 2 kb, 2.5 +/- 0.8 fold, Figure 3C). Transfection of Human ASM cells with BDKRB2 promoter constructs again identified the 1 kb and 4 kb promoter constructs as demonstrating the highest level of activity as shown at 20 hours post transfection (8.7 +/- 1.7 fold and 5.5 +/- 1.1 fold respectively, Figure 3D). pGL4-SV40-Luc2 control luciferase levels were; CHO-K1 (649 +/- 99 fold) and Human ASM (20 hours 1229 +/- 173 fold, 40 hours 1255 +/- 157 fold). These data confirmed that the primary ASM cells were being efficiently transfected using this protocol.


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)

Transcriptional activity of HRH1 and BDKRB2 core promoter fragments. Luciferase activity in Human ASM cell lysates 20 hours post transfection with control, HRH1 and BDKRB2 luciferase constructs (A and B) (n = 4), Luciferase activity in Human ASM cell lysates 40 hours post transfection with control, HRH1 and BDKRB2 luciferase constructs (C and D) (n = 5). Dunnett's Multiple Comparison Test (compared to pGL4-Luc2 control **p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Transcriptional activity of HRH1 and BDKRB2 core promoter fragments. Luciferase activity in Human ASM cell lysates 20 hours post transfection with control, HRH1 and BDKRB2 luciferase constructs (A and B) (n = 4), Luciferase activity in Human ASM cell lysates 40 hours post transfection with control, HRH1 and BDKRB2 luciferase constructs (C and D) (n = 5). Dunnett's Multiple Comparison Test (compared to pGL4-Luc2 control **p < 0.01).
Mentions: Preliminary luciferase experiments using CHO-K1 cells transfected with pGL4 constructs for 40 hours provided clear evidence that all of the HRH1 and BDKRB2 promoter-luciferase plasmids were transcriptionally active (all >15 fold over pGL4-Luc2 control, p = 0.0002 and p = 0.0004 respectively, data not shown). Interestingly the greatest activity in CHO-K1 cells was observed for the plasmids containing the longest segment of the two core promoters i.e. the HRH1-4 kb (106.5 +/- 34.3 fold over pGL4-Luc2, p < 0.01) and BDKRB2-4 kb (219.4 +/- 75.3 fold over pGL4-Luc2, p < 0.01) plasmids. Basal activity of HRH1 promoter containing plasmids transfected into Human ASM showed low level activity over pGL4-Luc2 at 20 hours post transfection with maximal activity observed for the pGL4-HRH1-2 kb transfection samples (2.1 +/- 0.2 fold, Figure 3A). Analyses of luciferase activity in Human ASM cells transfected with the series of BDKRB2 or control constructs for 20 hours demonstrated significant differences in activity between constructs (ANOVA, p = 0.0001). The BDKRB2 1 kb (5.6 +/- 0.9 fold) and 4 kb (3.8 +/- 0.8 fold) constructs showed clear activity (Figure 3B). The pGL4-BDKRB2-3 kb transfection samples showed similar to pGL4-Luc2 activity (Figure 3B). Transfection of Human ASM cells with HRH1 promoter containing constructs for 40 hours again demonstrated a modest transcriptional activity over pGL4-Luc2 as observed for the 20 hour transfections (maximum HRH1 2 kb, 2.5 +/- 0.8 fold, Figure 3C). Transfection of Human ASM cells with BDKRB2 promoter constructs again identified the 1 kb and 4 kb promoter constructs as demonstrating the highest level of activity as shown at 20 hours post transfection (8.7 +/- 1.7 fold and 5.5 +/- 1.1 fold respectively, Figure 3D). pGL4-SV40-Luc2 control luciferase levels were; CHO-K1 (649 +/- 99 fold) and Human ASM (20 hours 1229 +/- 173 fold, 40 hours 1255 +/- 157 fold). These data confirmed that the primary ASM cells were being efficiently transfected using this protocol.

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