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Investigating the Role of the G-proteins Gq and G11 in Smooth Muscle Cell Contraction-Induced Remodelling in Asthma
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A7268 - Investigating the Role of the G-proteins Gq and G11 in Smooth Muscle Cell Contraction-Induced Remodelling in Asthma
Author Block: R. J. Middlewick, D. Shaw, A. L. Tatler, R. G. Jenkins; Respiratory Medicine, University of Nottingham, Nottingham, United Kingdom.
Rationale: Asthma is a chronic inflammatory disease of the airways affecting 300 million individuals worldwide. Asthma pathology is characterised by airway hyper-responsiveness, bronchoconstriction and, particularly in severe asthma, airway remodelling. Inflammation was thought to be the main driver of airway remodelling, however recent evidence suggests that bronchoconstriction is central to this process. The pleotropic cytokine Transforming Growth Factor beta (TGFβ) can mediate many of the structural changes associated with airway remodelling. TGFβ is activated via an integrin mediated pathway upon contraction of airway smooth muscle (ASM) cells and drives the transcription of remodelling genes. The G proteins Gq and G11 (Gq/11) regulate integrin-mediated TGFβ activation in lung epithelial cells, however, their role in bronchoconstriction-induced TGFβ activation and therefore airway remodelling is unknown. Here the role of the heterotrimeric G-proteins Gq/11 in bronchoconstriction agonist-induced TGFβ activation is explored. Methods: Transformed mink lung epithelial cells (TMLC) containing a TGFβ responsive portion of the PAI1 promotor driving luciferase expression were used as a TGFβ reporter cell. TMLC in coculture with ASM cells (passage 6) isolated from asthmatic (n=4) or non-asthmatic (n=4) were treated with the contractile agonist lysophosphatidic acid (LPA). To investigate the role of Gq/11 the inhibitor YM-254890 was used, which specifically inhibits Gq/11 and G14. To determine the effect of inhibiting Gq/11 and G14 on expression of airway remodelling genes quantitative RT-PCR was utilised. Results: Coculture assay of asthmatic and non-asthmatic ASM cells showed TGFβ activation occurred in a dose-dependent manner to LPA stimulation that was increased at the top concentration in every cell line. The Gq/11 and G14 inhibitor YM-254890 inhibited LPA-induced TGFβ in a concentration-dependent manner that was maximal at 10nM (p=0.002). YM-254890 had no effect on TMLC cells alone. Furthermore, YM-254890 inhibited LPA-induced PAI1 mRNA expression after 4 hours of 100uM LPA stimulation in asthmatic ASM cells (n=2). Conclusions: The heterotrimeric G proteins Gq/11 are required for LPA-induced TGFβ activation in ASM cells. This raises the intriguing possibility that inhibition of Gq/11 may protect against bronchoconstriction-induced airway remodelling driven by TGFβ in severe asthma.
Author Block: R. J. Middlewick, D. Shaw, A. L. Tatler, R. G. Jenkins; Respiratory Medicine, University of Nottingham, Nottingham, United Kingdom.
Rationale: Asthma is a chronic inflammatory disease of the airways affecting 300 million individuals worldwide. Asthma pathology is characterised by airway hyper-responsiveness, bronchoconstriction and, particularly in severe asthma, airway remodelling. Inflammation was thought to be the main driver of airway remodelling, however recent evidence suggests that bronchoconstriction is central to this process. The pleotropic cytokine Transforming Growth Factor beta (TGFβ) can mediate many of the structural changes associated with airway remodelling. TGFβ is activated via an integrin mediated pathway upon contraction of airway smooth muscle (ASM) cells and drives the transcription of remodelling genes. The G proteins Gq and G11 (Gq/11) regulate integrin-mediated TGFβ activation in lung epithelial cells, however, their role in bronchoconstriction-induced TGFβ activation and therefore airway remodelling is unknown. Here the role of the heterotrimeric G-proteins Gq/11 in bronchoconstriction agonist-induced TGFβ activation is explored. Methods: Transformed mink lung epithelial cells (TMLC) containing a TGFβ responsive portion of the PAI1 promotor driving luciferase expression were used as a TGFβ reporter cell. TMLC in coculture with ASM cells (passage 6) isolated from asthmatic (n=4) or non-asthmatic (n=4) were treated with the contractile agonist lysophosphatidic acid (LPA). To investigate the role of Gq/11 the inhibitor YM-254890 was used, which specifically inhibits Gq/11 and G14. To determine the effect of inhibiting Gq/11 and G14 on expression of airway remodelling genes quantitative RT-PCR was utilised. Results: Coculture assay of asthmatic and non-asthmatic ASM cells showed TGFβ activation occurred in a dose-dependent manner to LPA stimulation that was increased at the top concentration in every cell line. The Gq/11 and G14 inhibitor YM-254890 inhibited LPA-induced TGFβ in a concentration-dependent manner that was maximal at 10nM (p=0.002). YM-254890 had no effect on TMLC cells alone. Furthermore, YM-254890 inhibited LPA-induced PAI1 mRNA expression after 4 hours of 100uM LPA stimulation in asthmatic ASM cells (n=2). Conclusions: The heterotrimeric G proteins Gq/11 are required for LPA-induced TGFβ activation in ASM cells. This raises the intriguing possibility that inhibition of Gq/11 may protect against bronchoconstriction-induced airway remodelling driven by TGFβ in severe asthma.
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