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ABSTRACT: Acetylcholine has traditionally only been regarded as a neurotransmitter of the parasympathetic nervous system, causing bronchoconstriction and mucus secretion in asthma and COPD by muscarinic receptor activation on airway smooth muscle and mucus-producing cells. Recent studies in experimental models indicate that muscarinic receptor stimulation in the airways also induces pro-inflammatory, pro-proliferative and pro-fibrotic effects, which may involve activation of airway structural and inflammatory cells by neuronal as well as non-neuronal acetylcholine. In addition, mechanical changes caused by muscarinic agonist-induced bronchoconstriction may be involved in airway remodeling. Crosstalk between muscarinic receptors and β2-adrenoceptors on airway smooth muscle causes a reduced bronchodilator response to β2-agonists, and a similar mechanism could possibly apply to the poor inhibition of inflammatory and remodeling processes by these drugs. Collectively, these findings provide novel perspectives for muscarinic receptor antagonists in asthma and COPD, since these drugs may not only acutely affect cholinergic airways obstruction, but also have important beneficial effects on β2-agonist responsiveness, airway inflammation and remodeling. The clinical relevance of these findings is presently under investigation and starting to emerge.
Current Opinion in Pharmacology 05/2013; · 6.86 Impact Factor
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ABSTRACT: Transforming growth factor-β1 (TGF-β1) is a central mediator in tissue remodelling processes, including fibrosis and airway smooth muscle (ASM) hyperplasia as observed in asthma. The mechanisms underlying this response, however, are unclear as TGF-β1 has only weak mitogenic effects on ASM cells. In this study we hypothesized that the mitogenic effect of TGF-β1 on ASM is indirect and requires prolonged exposure to allow extracellular matrix (ECM) deposition. To address this hypothesis, we investigated the effects of acute and prolonged treatment with TGF-β1, alone and in combination with the muscarinic receptor agonist methacholine on human ASM cell proliferation. Acutely, TGF-β1 had no mitogenic effect; however, prolonged treatment (7 days) with TGF-β1 increased ASM cell proliferation and potentiated the PDGF-induced mitogenic response. Muscarinic receptor stimulation with methacholine synergistically enhanced the effect of TGF-β1. Interestingly, the integrin-blocking peptide RGDS (Arg-Gly-Asp-Ser) as well as integrin α5β1 function-blocking antibodies inhibited the effects of TGF-β1 and its combination with methacholine on cell proliferation. Accordingly, prolonged treatment with TGF-β1 increased fibronectin expression, which was also synergistically enhanced by methacholine. The synergistic effects of methacholine on TGF-β1-induced proliferation were reduced by the long-acting muscarinic receptor antagonist tiotropium and the M2 receptor subtype selective antagonist gallamine, but not the M3 selective antagonist DAU5884. In line with these findings, the irreversible Gi-protein inhibitor pertussis toxin also prevented the potentiation of TGF-β1-induced proliferation by methacholine. We conclude that prolonged exposure to TGF-β1 enhances ASM cell proliferation, which is mediated by ECM-integrin interactions and can be enhanced by muscarinic M2 receptor stimulation.
American Journal of Respiratory Cell and Molecular Biology 02/2013; · 5.13 Impact Factor
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ABSTRACT: BACKGROUND: WNT signalling is activated during lung tissue damage and inflammation. We investigated whether lung epithelial expression of WNT ligands, receptors (frizzled; FZD) or target genes is dysregulated on cigarette smoking and/or in chronic obstructive pulmonary disease (COPD). METHODS: We studied this in human lung epithelial cell lines and primary bronchial epithelial cells (PBEC) from COPD patients and control (non-)smokers, at baseline and on cigarette smoke extract (CSE) exposure. RESULTS: CSE significantly decreased WNT-4, WNT-10B and FZD2 and increased WNT-5B mRNA expression in 16HBE, but did not affect WNT-4 protein. The mRNA expression of WNT-4, but not other WNT ligands, was lower in PBEC from smokers than non-smokers and downregulated by CSE in PBEC from all groups, yet higher in PBEC from COPD patients than control smokers. Moreover, PBEC from COPD patients displayed higher WNT-4 protein expression than both smokers and non-smokers. Exogenously added WNT-4 significantly increased CXCL8/IL-8, IL-6, CCL5/RANTES, CCL2/MCP-1 and vascular endothelial growth factor (VEGF) secretion in 16HBE, but did not affect the canonical WNT target genes MMP-2, MMP-9, fibronectin, β-catenin, Dickkopf and axin-2, and induced activation of the non-canonical signalling molecule p38. Moreover, WNT-4 potentiated the CSE-induced upregulation of IL-8 and VEGF. CONCLUSIONS: WNT-4 mRNA and protein levels are higher in PBEC from COPD patients than control (non-)smokers, while cigarette smoke downregulates airway epithelial WNT-4 mRNA, but not protein expression. As WNT-4 further increases CSE-induced pro-inflammatory cytokine release in bronchial epithelium, we propose that higher epithelial WNT-4 levels in combination with cigarette smoking may have important implications for the development of airway inflammation in COPD.
Thorax 01/2013; · 6.84 Impact Factor
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ABSTRACT: BACKGROUND: Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. Transforming growth factor-β (TGF-β) is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signaling pathways; its role in TGF-β(1) -induced myofibroblast differentiation is currently largely unknown. PURPOSE: To determine the contribution of GSK-3 signaling in TGF-β(1) -induced myofibroblast differentiation. EXPERIMENTAL APPROACH: We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis, respectively. RESULTS: Stimulation of MRC5 and primary human lung fibroblasts with TGF-β(1) resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-β(1) -induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-β(1) -induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB, nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as functional antagonist of TGF-β/smad signaling. CONCLUSION AND IMPLICATION: We demonstrate that GSK-3 signaling regulates TGF-β(1) -induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases.
British Journal of Pharmacology 01/2013; · 4.41 Impact Factor
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Kuldeep Kumawat,
Mark H Menzen,
I Sophie T Bos,
Hoeke A Baarsma,
Pieter Borger,
Michael Roth,
Michael Tamm,
Andrew J Halayko,
Mirjam Simoons,
Alita Prins,
Dirkje S Postma,
Martina Schmidt, Reinoud Gosens
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ABSTRACT: Transforming growth factor β (TGF-β), a key mediator of fibrotic responses, is increased in asthma and drives airway remodeling by inducing expression of extracellular matrix (ECM) proteins. We investigated the molecular mechanisms underlying TGF-β-induced ECM expression by airway smooth muscle cells and demonstrate a novel link between TGF-β and Wingless/integrase 1 (WNT) signaling in ECM deposition. Airway smooth muscle expresses abundant WNT ligands, with the noncanonical WNT-5A being the most profoundly expressed. Interestingly, WNT-5A shows ∼2-fold higher abundance in airway smooth muscle cells isolated from individuals with asthma than individuals without asthma. WNT-5A is markedly induced in response to TGF-β (4-16-fold; EC(50) 0.3 ng/ml) and is required for collagen and fibronectin expression by airway smooth muscle. WNT-5A engages noncanonical WNT signaling pathways, as inhibition of Ca(2+) and c-Jun N-terminal kinase (JNK) signaling attenuated this TGF-β response, whereas the canonical WNT antagonist Dickkopf 1 (DKK-1) did not. Accordingly, WNT-5A induced JNK phosphorylation and nuclear translocation of nuclear factor of activated T cells c1 (NFATc1). Furthermore, silencing of the WNT-5A receptors Frizzled 8 (FZD(8)) and RYK attenuated TGF-β-induced ECM expression. Collectively, these findings demonstrate that noncanonical WNT-5A signaling is activated by and necessary for TGF-β-induced ECM production by airway smooth muscle cells, which could have significance in asthma pathogenesis.-Kumawat, K., Menzen, M. H.., Bos, I. S. T., Baarsma, H. A., Borger, P., Roth, M., Tamm, M., Halayko, A. J., Simoons, M., Prins, A., Postma, D. S., Schmidt, M., and Gosens, R. Noncanonical WNT-5A signaling regulates TGF-β-induced extracellular matrix production by airway smooth muscle cells.
The FASEB Journal 12/2012; · 5.71 Impact Factor
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ABSTRACT: Since ancient times, anticholinergics have been used as a bronchodilator therapy for obstructive lung diseases. Targets of these drugs are G-protein-coupled muscarinic M(1), M(2) and M(3) receptors in the airways, which have long been recognized to regulate vagally-induced airway smooth muscle contraction and mucus secretion. However, recent studies have revealed that acetylcholine also exerts pro-inflammatory, pro-proliferative and pro-fibrotic actions in the airways, which may involve muscarinic receptor stimulation on mesenchymal, epithelial and inflammatory cells. Moreover, acetylcholine in the airways may not only be derived from vagal nerves, but also from non-neuronal cells, including epithelial and inflammatory cells. Airway smooth muscle cells seem to play a major role in the effects of acetylcholine on airway function. It has become apparent that these cells are multipotent cells that may reversibly adopt (hyper)contractile, proliferative and synthetic phenotypes, which are all under control of muscarinic receptors and differentially involved in bronchoconstriction, airway remodeling and inflammation. Cholinergic contractile tone is increased by airway inflammation associated with asthma and COPD, resulting from exaggerated acetylcholine release as well as increased expression of contraction related proteins in airway smooth muscle. Moreover, muscarinic receptor stimulation promotes proliferation of airway smooth muscle cells as well as fibroblasts, and regulates cytokine, chemokine and extracellular matrix production by these cells, which may contribute to airway smooth muscle growth, airway fibrosis and inflammation. In line, animal models of chronic allergic asthma and COPD have recently demonstrated that tiotropium may potently inhibit airway inflammation and remodeling. These observations indicate that muscarinic receptors have a much larger role in the pathophysiology of obstructive airway diseases than previously thought, which may have important therapeutic implications.
Pulmonary Pharmacology & Therapeutics 07/2012; · 2.80 Impact Factor
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ABSTRACT: Acetylcholine is the primary parasympathetic neurotransmitter in the airways and an autocrine/paracrine secreted hormone from non-neuronal origins including inflammatory cells and airway structural cells. In addition to the well-known functions of acetylcholine in regulating bronchoconstriction and mucus secretion, it is increasingly evident that acetylcholine regulates inflammatory cell chemotaxis and activation, and also participates in signaling events leading to chronic airway wall remodeling that is associated with chronic obstructive airway diseases including asthma and COPD. As muscarinic receptors appear responsible for most of the pro-inflammatory and remodeling effects of acetylcholine, these findings have significant implications for anticholinergic therapy in asthma and COPD, which is selective for muscarinic receptors. Here, the regulatory role of acetylcholine in inflammation and remodeling in asthma and COPD will be discussed including the perspectives that these findings offer for anticholinergic therapy in these diseases.
Life sciences 03/2012; · 2.56 Impact Factor
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ABSTRACT: Acetylcholine is the predominant parasympathetic neurotransmitter in the airways that regulates bronchoconstriction and mucus secretion. Recent findings suggest that acetylcholine regulates additional functions in the airways, including inflammation and remodelling during inflammatory airway diseases. Moreover, it has become apparent that acetylcholine is synthesized by nonneuronal cells and tissues, including inflammatory cells and structural cells. In this paper, we will discuss the regulatory role of acetylcholine in inflammation and remodelling in which we will focus on the role of the airway smooth muscle cell as a target cell for acetylcholine that modulates inflammation and remodelling during respiratory diseases such as asthma and COPD.
Journal of Allergy 01/2012; 2012:681258.
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ABSTRACT: Chronic inflammatory airway diseases like asthma and chronic obstructive pulmonary disease (COPD) are characterized by airway remodeling with altered extracellular matrix (ECM) deposition. Transforming growth factor-β(1) (TGF-β(1)) is upregulated in asthma and COPD and contributes to tissue remodeling in the airways by driving ECM production by structural cells, including airway smooth muscle. In this study, we investigated the activation of β-catenin signaling and its contribution to ECM production by airway smooth muscle cells in response to TGF-β(1). Stimulation of airway smooth muscle cells with TGF-β(1) resulted in a time-dependent increase of total and nonphosphorylated β-catenin protein expression via induction of β-catenin mRNA and inhibition of GSK-3. In addition, the TGF-β(1)-induced β-catenin activated TCF/LEF-dependent gene transcription, as determined by the β-catenin sensitive TOP-flash luciferase reporter assay. Furthermore, TGF-β(1) stimulation increased mRNA expression of collagen Iα1, fibronectin, versican, and PAI-1. Pharmacological inhibition of β-catenin by PKF115-584 or downregulation of β-catenin expression by specific small interfering RNA (siRNA) substantially inhibited TGF-β(1)-induced expression of the ECM genes. Fibronectin protein deposition by airway smooth muscle cells in response to TGF-β(1) was also inhibited by PKF115-584 and β-catenin siRNA. Moreover, transfection of airway smooth muscle cells with a nondegradable β-catenin mutant (S33Y β-catenin) was sufficient for inducing fibronectin protein expression. Collectively, these findings indicate that β-catenin signaling is activated in response to TGF-β(1) in airway smooth muscle cells, which is required and sufficient for the regulation of ECM protein production. Targeting β-catenin-dependent gene transcription may therefore hold promise as a therapeutic intervention in airway remodeling in both asthma and COPD.
AJP Lung Cellular and Molecular Physiology 09/2011; 301(6):L956-65. · 3.66 Impact Factor
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ABSTRACT: One of the pathways activated during liver fibrosis is the Rho kinase pathway, which regulates activation, migration, and contraction of hepatic stellate cells (HSC). Inhibition of this kinase by the Rho kinase inhibitor Y27632 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride] has been shown to reduce fibrosis in animal models. However, kinase expression is ubiquitous, so any inhibitor may affect many cell types. We hypothesize that cell-specific delivery of a kinase inhibitor will be beneficial. Therefore, we conjugated Y27632 to the carrier mannose-6-phosphate (M6P) human serum albumin (HSA), which is taken up specifically in activated HSC through the M6P/insulin-like growth factor II receptor. This conjugate decreased protein expression of phosphorylated myosin light chain 2 (pMLC2) and vinculin, downstream of Rho kinase, in activated primary HSC and decreased the migration and contraction of HSC. In an ex vivo model, free Y27632 decreased contractility of rat aortas, whereas the Y27-conjugate did not, showing that the Y27-conjugate does not affect nontarget tissue. In chronic CCl(4)-induced liver fibrosis, both free drug and conjugate reduced HSC activation; however, only the Y27-conjugate significantly reduced collagen deposition. Treatment with the Y27-conjugate, but not with free drug, reduced pMLC2 expression in livers 24 h after injection, demonstrating prolonged inhibition of the Rho kinase pathway. The Rho kinase inhibitor Y27632 can be specifically targeted to HSC using M6PHSA, decreasing its effects in nontarget tissues. The targeted drug effectively reduced fibrotic parameters in vivo via the inhibition of the Rho kinase pathway.
Journal of Pharmacology and Experimental Therapeutics 03/2011; 337(3):628-35. · 3.83 Impact Factor
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ABSTRACT: Glycogen synthase kinase-3 (GSK-3) is a constitutively active kinase that regulates multiple signaling proteins and transcription factors involved in inflammation. Its role in inflammatory lung diseases, including chronic obstructive pulmonary disease (COPD), is largely unknown. We investigated the role of GSK-3 in the secretion of chemokines and growth factors by human airway smooth muscle cells after exposure to cigarette smoke extract (CSE) or interleukin-1β (IL-1β), important factors involved in the development of COPD. Cultured human airway smooth muscle cells were exposed to increasing concentrations of CSE (1-15%) and IL-1β (0.01-1.0 ng/ml), which induced the secretion of VEGF-A and IL-8, whereas eotaxin secretion was induced by IL-1β only. Inhibition of GSK-3 by the selective inhibitor SB216763 or CHIR/CT99021 attenuated the cytokine and growth factor release induced by CSE and/or IL-1β, without affecting the basal release. Secretion of the cytokines by airway smooth muscle partially depends on NF-κB signaling, and GSK-3 has been implicated in regulating multiple steps in activating the NF-κB signaling pathway. IL-1β treatment induced degradation of the NF-κB inhibitory protein Iκ-Bα followed by nuclear translocation and DNA binding of p65 NF-κB, which were unaffected by inhibition of GSK-3. However, induction of NF-κB-dependent transcriptional activity by IL-1β and CSE was largely reduced upon GSK-3 inhibition by SB216763. Collectively, we demonstrate that CSE and IL-1β activate airway smooth muscle cells to secrete the proinflammatory cytokines IL-8, eotaxin, and VEGF-A. Furthermore, we show that GSK-3 regulates the release of these cytokines induced by CSE and IL-1β by promoting NF-κB-dependent gene transcription.
AJP Lung Cellular and Molecular Physiology 03/2011; 300(6):L910-9. · 3.66 Impact Factor
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ABSTRACT: Preparations of ivy leaves dry extract with secretolytic and bronchiolytic efficacy are widely used for the treatment of acute and chronic obstructive airway diseases. The mechanism by which ivy preparations improve lung functions is not fully understood. Here, we tested the influence of the three main saponins of ivy, α-hederin, hederacoside C and hederagenin, on the contraction and relaxation behaviour of isolated bovine tracheal smooth muscle strips by isometric tension measurements. None of the tested compounds altered histamine or methacholine-induced contraction of the smooth muscle strips. In contrast, the isoprenaline-induced relaxation of 100μM methacholine precontracted muscle strips was significantly enhanced when pre-treated with 1μM of α-hederin for 18h. The pre-treatment with hederacoside C or hederagenin had no effect on isoprenaline-induced relaxation. For the first time the bronchiolytic effect of α-hederin was demonstrated by isometric tension measurements using bovine tracheal smooth muscle strips. α-Hederin increases isoprenaline-induced relaxation indirectly, probably by inhibiting heterologous desensitization induced by high concentrations of muscarinic ligands like methacholine.
Phytomedicine: international journal of phytotherapy and phytopharmacology 01/2011; 18(2-3):214-8. · 2.17 Impact Factor
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ABSTRACT: Chronic obstructive pulmonary disease (COPD) is characterized by abnormal extracellular matrix (ECM) turnover. Recently, activation of the WNT/β-catenin pathway has been associated with abnormal ECM turnover in various chronic diseases. We determined WNT-pathway gene expression in pulmonary fibroblasts of individuals with and without COPD and disentangled the role of β-catenin in fibroblast phenotype and function.
We assessed the expression of WNT-pathway genes and the functional role of β-catenin, using MRC-5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD.
Pulmonary fibroblasts expressed mRNA of genes required for WNT signaling. Stimulation of fibroblasts with TGF-β₁, a growth factor important in COPD pathogenesis, induced WNT-5B, FZD₈, DVL3 and β-catenin mRNA expression. The induction of WNT-5B, FZD₆, FZD₈ and DVL3 mRNA by TGF-β₁ was higher in fibroblasts of individuals with COPD than without COPD, whilst basal expression was similar. Accordingly, TGF-β₁ activated β-catenin signaling, as shown by an increase in transcriptionally active and total β-catenin protein expression. Furthermore, TGF-β₁induced the expression of collagen1α1, α-sm-actin and fibronectin, which was attenuated by β-catenin specific siRNA and by pharmacological inhibition of β-catenin, whereas the TGF-β₁-induced expression of PAI-1 was not affected. The induction of transcriptionally active β-catenin and subsequent fibronectin deposition induced by TGF-β₁ were enhanced in pulmonary fibroblasts from individuals with COPD.
β-catenin signaling contributes to ECM production by pulmonary fibroblasts and contributes to myofibroblasts differentiation. WNT/β-catenin pathway expression and activation by TGF-β₁ is enhanced in pulmonary fibroblasts from individuals with COPD. This suggests an important role of the WNT/β-catenin pathway in regulating fibroblast phenotype and function in COPD.
PLoS ONE 01/2011; 6(9):e25450. · 4.09 Impact Factor
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Reinoud Gosens,
Gerald L Stelmack,
Sophie T Bos,
Gordon Dueck,
Mark M Mutawe,
Dedmer Schaafsma,
Helmut Unruh,
William T Gerthoffer,
Johan Zaagsma,
Herman Meurs,
Andrew J Halayko
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ABSTRACT: Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β(1). As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β(1) induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β(1). The failure by TGF-β(1) to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β(1) signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.
Journal of Cellular and Molecular Medicine 12/2010; 15(11):2430-42. · 4.13 Impact Factor
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ABSTRACT: beta-Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for beta-catenin in the regulation of smooth muscle force production. beta-Catenin colocalized with smooth muscle alpha-actin (sm-alpha-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-alpha-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-alpha-actin and beta-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological beta-catenin/T cell factor-4 (TCF4) inhibitor PKF115-584 (100 nM) reduced beta-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KCl- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-alpha-actin or sm-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of beta-catenin in BTSM strips reduced beta-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-alpha-actin or sm-MHC expression. Conversely, pharmacological (SB-216763, LiCl) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of beta-catenin and augmented maximal KCl- and methacholine-induced contractions. We conclude that beta-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells.
AJP Lung Cellular and Molecular Physiology 08/2010; 299(2):L204-14. · 3.66 Impact Factor
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ABSTRACT: Abstract
Background
A major feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, which includes an increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodelling in COPD are currently unknown. We hypothesized that cigarette smoke (CS) and/or lipopolysaccharide (LPS), a major constituent of CS, organic dust and gram-negative bacteria, that may be involved in recurrent airway infections and exacerbations in COPD patients, would induce phenotype changes of ASM.
Methods
To this aim, using cultured bovine tracheal smooth muscle (BTSM) cells and tissue, we investigated the direct effects of CS extract (CSE) and LPS on ASM proliferation and contractility.
Results
Both CSE and LPS induced a profound and concentration-dependent increase in DNA synthesis in BTSM cells. CSE and LPS also induced a significant increase in BTSM cell number, which was associated with increased cyclin D1 expression and dependent on activation of ERK 1/2 and p38 MAP kinase. Consistent with a shift to a more proliferative phenotype, prolonged treatment of BTSM strips with CSE or LPS significantly decreased maximal methacholine- and KCl-induced contraction.
Conclusions
Direct exposure of ASM to CSE or LPS causes the induction of a proliferative, hypocontractile ASM phenotype, which may be involved in airway remodelling in COPD.
Respiratory Research. 01/2010;
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ABSTRACT: A major feature of chronic obstructive pulmonary disease (COPD) is airway remodelling, which includes an increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodelling in COPD are currently unknown. We hypothesized that cigarette smoke (CS) and/or lipopolysaccharide (LPS), a major constituent of CS, organic dust and gram-negative bacteria, that may be involved in recurrent airway infections and exacerbations in COPD patients, would induce phenotype changes of ASM.
To this aim, using cultured bovine tracheal smooth muscle (BTSM) cells and tissue, we investigated the direct effects of CS extract (CSE) and LPS on ASM proliferation and contractility.
Both CSE and LPS induced a profound and concentration-dependent increase in DNA synthesis in BTSM cells. CSE and LPS also induced a significant increase in BTSM cell number, which was associated with increased cyclin D1 expression and dependent on activation of ERK 1/2 and p38 MAP kinase. Consistent with a shift to a more proliferative phenotype, prolonged treatment of BTSM strips with CSE or LPS significantly decreased maximal methacholine- and KCl-induced contraction.
Direct exposure of ASM to CSE or LPS causes the induction of a proliferative, hypocontractile ASM phenotype, which may be involved in airway remodelling in COPD.
Respiratory research 01/2010; 11:48. · 3.36 Impact Factor
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ABSTRACT: Abstract Background Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown. Methods The present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M2 and M3 receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1β. Results Muscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1β. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1β in hASMc. Conclusions We conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.
Respiratory Research. 01/2010;
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ABSTRACT: Acetylcholine, the primary parasympathetic neurotransmitter in the airways, plays an important role in bronchoconstriction and mucus production. Recently, it has been shown that acetylcholine, by acting on muscarinic receptors, is also involved in airway inflammation and remodelling. The mechanism(s) by which muscarinic receptors regulate inflammatory responses are, however, still unknown.
The present study was aimed at characterizing the effect of muscarinic receptor stimulation on cytokine secretion by human airway smooth muscle cells (hASMc) and to dissect the intracellular signalling mechanisms involved. hASMc expressing functional muscarinic M2 and M3 receptors were stimulated with the muscarinic receptor agonist methacholine, alone, and in combination with cigarette smoke extract (CSE), TNF-α, PDGF-AB or IL-1β.
Muscarinic receptor stimulation induced modest IL-8 secretion by itself, yet augmented IL-8 secretion in combination with CSE, TNF-α or PDGF-AB, but not with IL-1β. Pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely normalized the effect of methacholine on CSE-induced IL-8 secretion, whereas PMA, a PKC activator, mimicked the effects of methacholine, inducing IL-8 secretion and augmenting the effects of CSE. Similar inhibition was observed using inhibitors of IκB-kinase-2 (SC514) and MEK1/2 (U0126), both downstream effectors of PKC. Accordingly, western blot analysis revealed that methacholine augmented the degradation of IκBα and the phosphorylation of ERK1/2 in combination with CSE, but not with IL-1β in hASMc.
We conclude that muscarinic receptors facilitate CSE-induced IL-8 secretion by hASMc via PKC dependent activation of IκBα and ERK1/2. This mechanism could be of importance for COPD patients using anticholinergics.
Respiratory research 01/2010; 11:130. · 3.36 Impact Factor
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ABSTRACT: Airway remodeling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. The mechanisms driving these changes are, however, incompletely understood. Recently, an important role for extracellular matrix proteins in regulating ASM proliferation and contractility has been found, suggesting that matrix proteins and their integrins actively modulate airway remodeling.
To investigate the role of RGD (Arg-Gly-Asp)-binding integrins in airway remodeling in an animal model of allergic asthma.
Using a guinea pig model of allergic asthma, the effects of topical application of the integrin-blocking peptide RGDS (Arg-Gly-Asp-Ser) and its negative control GRADSP (Gly-Arg-Ala-Asp-Ser-Pro) were assessed on markers of ASM remodeling, fibrosis, and inflammation induced by repeated allergen challenge. In addition, effects of these peptides on human ASM proliferation and maturation were investigated in vitro.
RGDS attenuated allergen-induced ASM hyperplasia and hypercontractility as well as increased pulmonary expression of smooth muscle myosin heavy chain and the proliferative marker proliferating cell nuclear antigen (PCNA). No effects were observed for GRADSP. The RGDS effects were ASM selective, as allergen-induced eosinophil and neutrophil infiltration as well as fibrosis were unaffected. In cultured human ASM cells, we demonstrated that proliferation induced by collagen I, fibronectin, serum, and platelet-derived growth factor requires signaling via RGD-binding integrins, particularly of the alpha(5)beta(1) subtype. In addition, RGDS inhibited smooth muscle alpha-actin accumulation in serum-deprived ASM cells.
This is the first study indicating that integrins modulate ASM remodeling in an animal model of allergic asthma, which can be inhibited by a small peptide containing the RGD motif.
American Journal of Respiratory and Critical Care Medicine 12/2009; 181(6):556-65. · 11.08 Impact Factor
