Muscarinic receptor signaling in the pathophysiology of asthma and COPD

Anticholinergics are widely used for the treatment of COPD, and to a lesser extent for asthma. Primarily used as bronchodilators, they reverse the action of vagally derived acetylcholine on airway smooth muscle contraction. Recent novel studies suggest that the effects of anticholinergics likely extend far beyond inducing bronchodilation, as the novel anticholinergic drug tiotropium bromide can effectively inhibit accelerated decline of lung function in COPD patients. Vagal tone is increased in airway inflammation associated with asthma and COPD; this results from exaggerated acetylcholine release and enhanced expression of downstream signaling components in airway smooth muscle. Vagally derived acetylcholine also regulates mucus production in the airways. A number of recent research papers also indicate that acetylcholine, acting through muscarinic receptors, may in part regulate pathological changes associated with airway remodeling. Muscarinic receptor signalling regulates airway smooth muscle thickening and differentiation, both in vitro and in vivo. Furthermore, acetylcholine and its synthesizing enzyme, choline acetyl transferase (ChAT), are ubiquitously expressed throughout the airways. Most notably epithelial cells and inflammatory cells generate acetylcholine, and express functional muscarinic receptors. Interestingly, recent work indicates the expression and function of muscarinic receptors on neutrophils is increased in COPD. Considering the potential broad role for endogenous acetylcholine in airway biology, this review summarizes established and novel aspects of muscarinic receptor signaling in relation to the pathophysiology and treatment of asthma and COPD

Src mediates cytokine-stimulated gene expression in airway myocytes through ERK MAPK

The p38 and extracellular signal-regulated kinases (ERK) mitogen-activated protein kinases (MAPK) participate in cytokine-stimulated inflammatory gene expression in airway smooth muscle cells. The following study was undertaken to determine whether Src tyrosine kinases are signaling intermediaries upstream of cytokine-stimulated MAPK activation and gene expression. Treating human airway myocytes with interleukin (IL)-1β, tumor necrosis factor (TNF) α and interferon (IFN) γ caused a rapid 1.8-fold increase in Src family tyrosine kinase activity within 1 minute that remained 2.3 to 2.7 fold above basal conditions for 15 minutes. This activity was blocked by addition of 30 μM PP1, a pyrimidine inhibitor specific for Src family tyrosine kinases, in immune-complex assays to confirm that this stimulus activates Src tyrosine kinase. Addition of PP1 also blocked cytokine-stimulated expression of IL-1β, IL-6 and IL-8, while decreasing phosphorylation of ERK, but not p38 MAPK. Since this inflammatory stimulus may activate additional inflammatory signaling pathways downstream of Src, we tested the effects of PP1 on phosphorylation of signal transducers and activators of transcription (STAT). PP1 had no effect on cytokine-stimulated STAT 1 or STAT 3 phosphorylation. These results demonstrate that Src tyrosine kinases participate in the regulation of IL-1β, IL-6 and IL-8 expression and that these effects of Src are mediated through activation of ERK MAPK and not p38 MAPK or STAT1/STAT3 phosphorylation

The laminin β1-competing peptide YIGSR induces a hypercontractile, hypoproliferative airway smooth muscle phenotype in an animal model of allergic asthma

Background: Fibroproliferative airway remodelling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. In vitro studies have shown that maturation of ASM cells to a (hyper)contractile phenotype is dependent on laminin, which can be inhibited by the laminin-competing peptide Tyr-Ile-Gly-Ser-Arg (YIGSR). The role of laminins in ASM remodelling in chronic asthma in vivo, however, has not yet been established. Methods: Using an established guinea pig model of allergic asthma, we investigated the effects of topical treatment of the airways with YIGSR on features of airway remodelling induced by repeated allergen challenge, including ASM hyperplasia and hypercontractility, inflammation and fibrosis. Human ASM cells were used to investigate the direct effects of YIGSR on ASM proliferation in vitro. Results: Topical administration of YIGSR attenuated allergen-induced ASM hyperplasia and pulmonary expression of the proliferative marker proliferating cell nuclear antigen (PCNA). Treatment with YIGSR also increased both the expression of sm-MHC and ASM contractility in saline-and allergen-challenged animals; this suggests that treatment with the laminin-competing peptide YIGSR mimics rather than inhibits laminin function in vivo. In addition, treatment with YIGSR increased allergen-induced fibrosis and submucosal eosinophilia. Immobilized YIGSR concentration-dependently reduced PDGF-induced proliferation of cultured ASM to a similar extent as laminin-coated culture plates. Notably, the effects of both immobilized YIGSR and laminin were antagonized by soluble YIGSR. Conclusion: These results indicate that the laminin-competing peptide YIGSR promotes a contractile, hypoproliferative ASM phenotype in vivo, an effect that appears to be linked to the microenvironment in which the cells are exposed to the peptide

IL-9 Induces CCL11 Expression via STAT3 Signalling in Human Airway Smooth Muscle Cells

Background: Previous findings support the concept that IL-9 may play a significant role in mediating both pro-inflammatory and changes in airway responsiveness that characterizes the atopic asthmatic state. We previously demonstrated that human airway smooth muscle (ASM) cells express a functional IL-9R that mediate CCL11 expression. However, the signaling pathway governing this effect is not well understood. Methodology/Principal Findings: In this study, we showed that IL-9 mediated CCL11 expression in ASM cells does not rely on STAT6 or STAT5 but on STAT3 pathway. IL-9 induced rapid STAT3 activation in primary ASM cells that was not observed in case of STAT6 or STAT5. STAT3 binding to CCL11 promoter was also observed in vivo upon IL-9 stimulation of ASM cells. Disruption of STAT3 activity with SH2 domain binding inhibitory peptide results in significant reduction of IL-9 mediated CCL11 promoter activity. DN STAT3b over-expression in ASM cells, but not Ser 727 STAT3 or STAT6 DN, abolishes IL-9 mediated CCL11 promoter activity. Finally, STAT3 but not STAT6 silenced ASM cells showed significant reduction in IL-9 mediated CCL11 promoter activity and mRNA expression. Conclusion/Significance: Taken together, our results indicate that IL-9 mediated CCL11 via STAT3 signalling pathway ma

Regulation of actin dynamics by WNT-5A:implications for human airway smooth muscle contraction

A defining feature of asthma is airway hyperresponsiveness (AHR), which underlies the exaggerated bronchoconstriction response of asthmatics. The role of the airway smooth muscle (ASM) in AHR has garnered increasing interest over the years, but how asthmatic ASM differs from healthy ASM is still an active topic of debate. WNT-5A is increasingly expressed in asthmatic ASM and has been linked with Th2-high asthma. Due to its link with calcium and cytoskeletal remodelling, we propose that WNT-5A may modulate ASM contractility. We demonstrated that WNT-5A can increase maximum isometric tension in bovine tracheal smooth muscle strips. In addition, we show that WNT-5A is preferentially expressed in contractile human airway myocytes compared to proliferative cells, suggesting an active role in maintaining contractility. Furthermore, WNT-5A treatment drives actin polymerisation, but has no effect on intracellular calcium flux. Next, we demonstrated that WNT-5A directly regulates TGF-β1-induced expression of α-SMA via ROCK-mediated actin polymerization. These findings suggest that WNT-5A modulates fundamental mechanisms that affect ASM contraction and thus may be of relevance for AHR in asthma

Epac as a novel effector of airway smooth muscle relaxation

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)-elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP-mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre-contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine-induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine-induced RhoA activation, measured by both stress fibre formation and pull-down assay whereas the same Epac activation prevented methacholine-induced Rac1 inhibition measured by pull-down assay. Epac-driven inhibition of both methacholine-induced muscle contraction by Toxin B-1470, and MLC phosphorylation by the Rac1-inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac-mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre-contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases

The anti-proliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide

BACKBROUND: COPD is a common, highly debilitating disease of the airways, primarily caused by smoking. Chronic inflammation and structural remodelling are key pathological features of this disease caused, in part, by the aberrant function of airway smooth muscle (ASM). We have previously demonstrated that hydrogen sulfide (H2S) can inhibit ASM cell proliferation and CXCL8 release, from cells isolated from non-smokers. METHODS: We examined the effect of H2S upon ASM cells from COPD patients. ASM cells were isolated from non-smokers, smokers and patients with COPD (n = 9). Proliferation and cytokine release (IL-6 and CXCL8) of ASM was induced by FCS, and measured by bromodeoxyuridine incorporation and ELISA, respectively. RESULTS: Exposure of ASM to H2S donors inhibited FCS-induced proliferation and cytokine release, but was less effective upon COPD ASM cells compared to the non-smokers and smokers. The mRNA and protein expression of the enzymes responsible for endogenous H2S production (cystathionine-β-synthase [CBS] and 3-mercaptopyruvate sulphur transferase [MPST]) were inhibited by H2S donors. Finally, we report that exogenous H2S inhibited FCS-stimulated phosphorylation of ERK-1/2 and p38 mitogen activated protein kinases (MAPKs), in the non-smoker and smoker ASM cells, with little effect in COPD cells. CONCLUSIONS: H2S production provides a novel mechanism for the repression of ASM proliferation and cytokine release. The ability of COPD ASM cells to respond to H2S is attenuated in COPD ASM cells despite the presence of the enzymes responsible for H2S production

Proinflammatory and Th2 Cytokines Regulate the High Affinity IgE Receptor (FcεRI) and IgE-Dependant Activation of Human Airway Smooth Muscle Cells

BACKGROUND:The high affinity IgE receptor (FcepsilonRI) is a crucial structure for IgE-mediated allergic reactions. We have previously demonstrated that human airway smooth muscle (ASM) cells express the tetrameric (alphabetagamma2) FcepsilonRI, and its activation leads to marked transient increases in intracellular Ca(2+) concentration, release of Th-2 cytokines and eotaxin-1/CCL11. Therefore, it was of utmost importance to delineate the factors regulating the expression of FcepsilonRI in human (ASM) cells. METHODOLOGY/PRINCIPAL FINDINGS:Incubation of human bronchial and tracheal smooth muscle (B/TSM) cells with TNF-alpha, IL-1beta or IL-4 resulted in a significant increase in FcepsilonRI-alpha chain mRNA expression (p<0.05); and TNF-alpha, IL-4 enhanced the FcepsilonRI-alpha protein expression compared to the unstimulated control at 24, 72 hrs after stimulation. Interestingly, among all other cytokines, only TNF-alpha upregulated the FcepsilonRI-gamma mRNA expression. FcepsilonRI-gamma protein expression remained unchanged despite the nature of stimulation. Of note, as a functional consequence of FcepsilonRI upregulation, TNF-alpha pre-sensitization of B/TSM potentially augmented the CC (eotaxin-1/CCL11 and RANTES/CCL5, but not TARC/CCL17) and CXC (IL-8/CXCL8, IP-10/CXCL10) chemokines release following IgE stimulation (p<0.05, n = 3). Furthermore, IgE sensitization of B/TSM cells significantly enhanced the transcription of selective CC and CXC chemokines at promoter level compared to control, which was abolished by Lentivirus-mediated silencing of Syk expression. CONCLUSIONS/SIGNIFICANCE:Our data depict a critical role of B/TSM in allergic airway inflammation via potentially novel mechanisms involving proinflammatory, Th2 cytokines and IgE/FcepsilonRI complex

Disruption of AKAP-PKA Interaction Induces Hypercontractility With Concomitant Increase in Proliferation Markers in Human Airway Smooth Muscle

With the ability to switch between proliferative and contractile phenotype, airway smooth muscle (ASM) cells can contribute to the progression of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), in which airway obstruction is associated with ASM hypertrophy and hypercontractility. A-kinase anchoring proteins (AKAPs) have emerged as important regulatory molecules in various tissues, including ASM cells. AKAPs can anchor the regulatory subunits of protein kinase A (PKA), and guide cellular localization via various targeting domains. Here we investigated whether disruption of the AKAP-PKA interaction, by the cell permeable peptide stearated (st)-Ht31, alters human ASM proliferation and contractility. Treatment of human ASM with st-Ht31 enhanced the expression of protein markers associated with cell proliferation in both cultured cells and intact tissue, although this was not accompanied by an increase in cell viability or cell-cycle progression, suggesting that disruption of AKAP-PKA interaction on its own is not sufficient to drive ASM cell proliferation. Strikingly, st-Ht31 enhanced contractile force generation in human ASM tissue with concomitant upregulation of the contractile protein α-sm-actin. This upregulation of α-sm-actin was independent of mRNA stability, transcription or translation, but was dependent on proteasome function, as the proteasome inhibitor MG-132 prevented the st-Ht31 effect. Collectively, the AKAP-PKA interaction appears to regulate markers of the multi-functional capabilities of ASM, and this alter the physiological function, such as contractility, suggesting potential to contribute to the pathophysiology of airway diseases