Epithelial 3D-spheroids as a tool to study air pollutant-induced lung pathology.

Cigarette smoke (CS) and air pollutants (AP) activate pathological processes in bronchial epithelial cells resulting in lung function decline which severely impacts human health. Knowledge about the molecular mechanism(s) by which CS and AP induce pathology is limited. Our previous studies in 2D cultures of human bronchial epithelial (BEAS-2B) cells showed that CS exposure activates transforming growth factor-ß1 (TGF-ß1) release and signaling. Furthermore, CS exposure reduced the expression of E-cadherin, which was prevented by applying a TGF-ß1 neutralizing antibody. Exposure of BEAS-2B cells cultured in 2D to diesel exhaust particles (DEP) increased TGF-ß1 protein expression and reduced the expression of epithelial cell markers, whereas mesenchymal markers are upregulated. Conventional 2D cell culture may, however, not fully reflect the physiology of bronchial epithelial cells in vivo. To simulate the in vivo situation more closely we cultured the bronchial epithelial cells in a 3D environment in the current study. Treatment of epithelial spheroids with TGF-ß resulted in reduced E-cadherin and increased collagen I expression, indicating the activation of epithelial-to-mesenchymal transition (EMT). Similarly, exposure of spheroids to DEP induced and EMT-like phenotype. Collectively, our data indicate AP induces an EMT-like phenotype of BEAS-2B cells in 3D spheroid cultures. This opens new avenues for drug development for the treatment of lung diseases induced by AP. The 3D spheroid cell culture is a novel, innovative and physiologically relevant model for culturing a variety of cells. It is a versatile tool for both high-throughput studies and for identifying molecular mechanisms involved in bronchial epithelial cell (patho)physiology.

A drug screen with approved compounds identifies amlexanox as a novel Wnt/ß-catenin activator inducing lung epithelial organoid formation.

BACKGROUND AND PURPOSE: Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/ß-catenin signalling is reduced in emphysema, and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/ß-catenin signalling and assess its potential to activate lung epithelial cells and repair. EXPERIMENTAL APPROACH: We screened 1216 human-approved compounds for Wnt/ß-catenin signalling activation using luciferase reporter cells and selected candidates based on their computationally predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell-derived lung organoids and in vivo using a murine elastase-induced emphysema model. KEY RESULTS: The primary screen identified 16 compounds that significantly induced Wnt/ß-catenin-dependent luciferase activity. Selected compounds activated Wnt/ß-catenin signalling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/ß-catenin inhibition, confirming the Wnt/ß-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation, and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces pro-regenerative signalling in emphysema. CONCLUSION AND IMPLICATIONS: Using a drug screen based on Wnt/ß-catenin activity, organoid assays and a murine emphysema model, amlexanox was identified as a novel potential therapeutic agent for emphysema.

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.

Increased Extracellular Vesicles Mediate WNT5A Signaling in Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo)fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored.Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF.Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies.Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-ß in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A.Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.

The PDE4 inhibitor CHF-6001 and LAMAs inhibit bronchoconstriction-induced remodeling in lung slices.

Combination therapy of PDE4 inhibitors and anticholinergics induces bronchoprotection in COPD. Mechanical forces that arise during bronchoconstriction may contribute to airway remodeling. Therefore, we investigated the impact of PDE4 inhibitors and anticholinergics on bronchoconstriction-induced remodeling. Because of the different mechanism of action of PDE4 inhibitors and anticholinergics, we hypothesized functional interactions of these two drug classes. Guinea pig precision-cut lung slices were preincubated with the PDE4 inhibitors CHF-6001 or roflumilast and/or the anticholinergics tiotropium or glycopyorrolate, followed by stimulation with methacholine (10 µM) or TGF-ß1 (2 ng/ml) for 48 h. The inhibitory effects on airway smooth muscle remodeling, airway contraction, and TGF-ß release were investigated. Methacholine-induced protein expression of smooth muscle-myosin was fully inhibited by CHF-6001 (0.3-100 nM), whereas roflumilast (1 µM) had smaller effects. Tiotropium and glycopyrrolate fully inhibited methacholine-induced airway remodeling (0.1-30 nM). The combination of CHF-6001 and tiotropium or glycopyrrolate, in concentrations partially effective by themselves, fully inhibited methacholine-induced remodeling in combination. CHF-6001 did not affect airway closure and had limited effects on TGF-ß1-induced remodeling, but rather, it inhibited methacholine-induced TGF-ß release. The PDE4 inhibitor CHF-6001, and to a lesser extent roflumilast, and the LAMAs tiotropium and glycopyrrolate inhibit bronchoconstriction-induced remodeling. The combination of CHF-6001 and anticholinergics was more effective than the individual compounds. This cooperativity might be explained by the distinct mechanisms of action inhibiting TGF-ß release and bronchoconstriction.

Reduced Frizzled Receptor 4 Expression Prevents WNT/ß-Catenin-driven Alveolar Lung Repair in Chronic Obstructive Pulmonary Disease.

RATIONALE: Chronic obstructive pulmonary disease (COPD), in particular emphysema, is characterized by loss of parenchymal alveolar tissue and impaired tissue repair. Wingless and INT-1 (WNT)/ß-catenin signaling is reduced in COPD; however, the mechanisms thereof, specifically the role of the frizzled (FZD) family of WNT receptors, remain unexplored. OBJECTIVES: To identify and functionally characterize specific FZD receptors that control downstream WNT signaling in impaired lung repair in COPD. METHODS: FZD expression was analyzed in lung homogenates and alveolar epithelial type II (ATII) cells of never-smokers, smokers, patients with COPD, and two experimental COPD models by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence. The functional effects of cigarette smoke on FZD4, WNT/ß-catenin signaling, and elastogenic components were investigated in primary ATII cells in vitro and in three-dimensional lung tissue cultures ex vivo. Gain- and loss-of-function approaches were applied to determine the effects of FZD4 signaling on alveolar epithelial cell wound healing and repair, as well as on expression of elastogenic components. MEASUREMENTS AND MAIN RESULTS: FZD4 expression was reduced in human and experimental COPD lung tissues as well as in primary human ATII cells from patients with COPD. Cigarette smoke exposure down-regulated FZD4 expression in vitro and in vivo, along with reduced WNT/ß-catenin activity. Inhibition of FZD4 decreased WNT/ß-catenin-driven epithelial cell proliferation and wound closure, and it interfered with ATII-to-ATI cell transdifferentiation and organoid formation, which were augmented by FZD4 overexpression. Moreover, FZD4 restoration by overexpression or pharmacological induction led to induction of WNT/ß-catenin signaling and expression of elastogenic components in three-dimensional lung tissue cultures ex vivo. CONCLUSIONS: Reduced FZD4 expression in COPD contributes to impaired alveolar repair capacity.

Noncanonical WNT-5A signaling impairs endogenous lung repair in COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. One main pathological feature of COPD is the loss of functional alveolar tissue without adequate repair (emphysema), yet the underlying mechanisms are poorly defined. Reduced WNT-ß-catenin signaling is linked to impaired lung repair in COPD; however, the factors responsible for attenuating this pathway remain to be elucidated. Here, we identify a canonical to noncanonical WNT signaling shift contributing to COPD pathogenesis. We demonstrate enhanced expression of noncanonical WNT-5A in two experimental models of COPD and increased posttranslationally modified WNT-5A in human COPD tissue specimens. WNT-5A was increased in primary lung fibroblasts from COPD patients and induced by COPD-related stimuli, such as TGF-ß, cigarette smoke (CS), and cellular senescence. Functionally, mature WNT-5A attenuated canonical WNT-driven alveolar epithelial cell wound healing and transdifferentiation in vitro. Lung-specific WNT-5A overexpression exacerbated airspace enlargement in elastase-induced emphysema in vivo. Accordingly, inhibition of WNT-5A in vivo attenuated lung tissue destruction, improved lung function, and restored expression of ß-catenin-driven target genes and alveolar epithelial cell markers in the elastase, as well as in CS-induced models of COPD. We thus identify a novel essential mechanism involved in impaired mesenchymal-epithelial cross talk in COPD pathogenesis, which is amenable to therapy.

Age dictates a steroid-resistant cascade of Wnt5a, transglutaminase 2, and leukotrienes in inflamed airways.

BACKGROUND: Airway remodeling is a detrimental and refractory process showing age-dependent clinical manifestations that are mechanistically undefined. The leukotriene (LT) and wingless/integrase (Wnt) pathways have been implicated in remodeling, but age-specific expression profiles and common regulators remained elusive. OBJECTIVE: We sought to study the activation of the LT and Wnt pathways during early- or late-onset allergic airway inflammation and to address regulatory mechanisms and clinical relevance in normal human bronchial epithelial cells (NHBEs) and nasal polyp tissues. METHODS: Mice were sensitized with house dust mite (HDM) allergens from days 3, 15, or 60 after birth. Remodeling factors in murine bronchoalveolar lavage fluid, lung tissue, or human nasal polyp tissue were analyzed by means of Western blotting, immunoassays, or histology. Regulatory mechanisms were studied in cytokine/HDM-stimulated NHBEs and macrophages. RESULTS: Bronchoalveolar lavage fluid LT levels were increased in neonatal and adult but reduced in juvenile HDM-sensitized mice. Lungs of neonatally sensitized mice showed increased 5-lipoxygenase levels, whereas adult mice expressed more group 10 secretory phospholipase A2, Wnt5a, and transglutaminase 2 (Tgm2). Older mice showed colocalization of Wnt5a and LT enzymes in the epithelium, a pattern also observed in human nasal polyps. IL-4 promoted epithelial Wnt5a secretion, which upregulated macrophage Tgm2 expression, and Tgm2 inhibition in turn reduced LT release. Tgm2, group 10 secretory phospholipase A2, and LT enzymes in NHBEs and nasal polyps were refractory to corticosteroids. CONCLUSION: Our findings reveal age differences in LT and Wnt pathways during airway inflammation and identify a steroid-resistant cascade of Wnt5a, Tgm2, and LTs, which might represent a therapeutic target for airway inflammation and remodeling.

WNT Signaling in Lung Aging and Disease.

Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), represent a significant and increasing health burden. Current therapies are largely symptomatic, and novel therapeutic approaches are needed. Aging has emerged as a contributing factor for the development of both IPF and COPD because their prevalence increases with age, and several pathological features of these diseases resemble classical hallmarks of aging. Aging is thought to be driven in part by aberrant activity of developmental signaling pathways that thus might drive pathological changes, a process termed antagonistic pleiotropy or developmental drift. The developmental WNT pathway is fundamental for lung development, and altered WNT activity has been reported to contribute to the pathogenesis of CLD, in particular to COPD and IPF. Although to date only limited data on WNT signaling during lung aging exist, WNT signal regulation during aging and its effects on age-related pathologies in other organs have recently been investigated. In this review, we discuss evidence of dysregulated WNT signaling in CLD in the context of WNT signal alteration in organ aging and its potential impact on age-related cellular mechanisms, such as senescence or stem cell exhaustion.

Linking bronchopulmonary dysplasia to adult chronic lung diseases: role of WNT signaling.

Bronchopulmonary dysplasia (BPD) is one of the most common chronic lung diseases in infants caused by pre- and/or postnatal lung injury. BPD is characterized by arrested alveolarization and vascularization due to extracellular matrix remodeling, inflammation, and impaired growth factor signaling. WNT signaling is a critical pathway for normal lung development, and its altered signaling has been shown to be involved in the onset and progression of incurable chronic lung diseases in adulthood, such as chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis (IPF). In this review, we summarize the impact of WNT signaling on different stages of lung development and its potential contribution to developmental lung diseases, especially BPD, and chronic lung diseases in adulthood.

TGF-ß-induced profibrotic signaling is regulated in part by the WNT receptor Frizzled-8.

TGF-ß is important in lung injury and remodeling processes. TGF-ß and Wingless/integrase-1 (WNT) signaling are interconnected; however, the WNT ligand-receptor complexes involved are unknown. Thus, we aimed to identify Frizzled (FZD) receptors that mediate TGF-ß-induced profibrotic signaling. MRC-5 and primary human lung fibroblasts were stimulated with TGF-ß1, WNT-5A, or WNT-5B in the presence and absence of specific pathway inhibitors. Specific small interfering RNA was used to knock down FZD8. In vivo studies using bleomycin-induced lung fibrosis were performed in wild-type and FZD8-deficient mice. TGF-ß1 induced FZD8 specifically via Smad3-dependent signaling in MRC-5 and primary human lung fibroblasts. It is noteworthy that FZD8 knockdown reduced TGF-ß1-induced collagen Iα1, fibronectin, versican, α-smooth muscle (sm)-actin, and connective tissue growth factor. Moreover, bleomycin-induced lung fibrosis was attenuated in FZD8-deficient mice in vivo Although inhibition of canonical WNT signaling did not affect TGF-ß1-induced gene expression in vitro, noncanonical WNT-5B mimicked TGF-ß1-induced fibroblast activation. FZD8 knockdown reduced both WNT-5B-induced gene expression of fibronectin and α-sm-actin, as well as WNT-5B-induced changes in cellular impedance. Collectively, our findings demonstrate a role for FZD8 in TGF-ß-induced profibrotic signaling and imply that WNT-5B may be the ligand for FZD8 in these responses.-Spanjer, A. I. R., Baarsma, H. A., Oostenbrink, L. M., Jansen, S. R., Kuipers, C. C., Lindner, M., Postma, D. S., Meurs, H., Heijink, I. H., Gosens, R., Königshoff, M. TGF-ß-induced profibrotic signaling is regulated in part by the WNT receptor Frizzled-8.

miR-92a regulates TGF-ß1-induced WISP1 expression in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and fatal form of idiopathic interstitial pneumonia. MicroRNAs (miRNAs), short, single-stranded RNAs that regulate protein expression in a post-transcriptional manner, have recently been demonstrated to contribute to IPF pathogenesis. We have previously identified WNT1-inducible signaling pathway protein 1 (WISP1) as a highly expressed pro-fibrotic mediator in IPF, but the underlying mechanisms resulting in increased WISP1 expression, remain elusive. Here, we investigated whether WISP1 is a target of miRNA regulation. We applied a novel supervised machine learning approach, which predicted miR-30a/d and miR-92a target sites in regions of the human WISP1 3'UTR preferentially bound by the miRNA ribonucleoprotein complex. Both miRNAs were decreased in IPF samples, whereas WISP1 protein was increased. We demonstrated further that transforming growth factor (TGF)-ß1-induced WISP1 expression in primary lung fibroblasts in vitro and lung homogenates in vivo. Notably, miR-30a and miR-92a reversed TGF-ß1-induced WISP1 mRNA expression in lung fibroblasts. Moreover, miR-92a inhibition increased WISP1 protein expression in lung fibroblasts. An inverse relationship for WISP1 and miR-92a was found in a TGF-ß1 dependent lung fibrosis model in vivo. Finally, we found significantly increased WISP1 expression in primary IPF fibroblasts, which negatively correlated with miR-92a level ex vivo. Altogether, our findings indicate a regulatory role of miR-92a for WISP1 expression in pulmonary fibrosis.

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy. METHODS: Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors. RESULTS: Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors. CONCLUSIONS: In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a constitutively active kinase that regulates multiple signalling proteins and transcription factors involved in a myriad of cellular processes. The kinase acts as a negative regulator in ß-catenin signalling and is critically involved in the smad pathway. Activation of both pathways may contribute to pulmonary features of chronic obstructive pulmonary disease (COPD). METHODS: In the present study, we investigated the effect of the selective GSK-3 inhibitor SB216763 on pulmonary pathology in a guinea pig model of lipopolysaccharide (LPS)-induced COPD. Guinea pigs were instilled intranasally with LPS or saline twice weekly for 12 weeks and pre-treated with either intranasally instilled SB216763 or corresponding vehicle 30 min prior to each LPS/saline challenge. RESULTS: Repeated LPS exposures activated ß-catenin signalling, primarily in the airway epithelium and submucosa. LPS also induced pulmonary inflammation and tissue remodelling as indicated by inflammatory cell influx, increased pulmonary fibronectin expression and enhanced small airway collagen content. Inhibition of GSK-3 by SB216763 did not affect LPS-induced inflammatory cell influx, but prevented the small airway remodelling and, unexpectedly, inhibited the activation of ß-catenin in vivo. LPS or SB216763 treatment had no effect on the airway smooth muscle content and alveolar airspace size. However, GSK-3 inhibition prevented LPS-induced right ventricle hypertrophy. CONCLUSIONS: Our findings indicate that GSK-3 inhibition prevents LPS-induced pulmonary pathology in guinea pigs, and that locally reduced LPS-induced ß-catenin activation appears in part to underlie this effect.

Glycogen synthase kinase-3 (GSK-3) regulates TGF-ß1-induced differentiation of pulmonary fibroblasts.

BACKGROUND: Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-ß 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 signalling pathways; its role in TGF-ß1-induced myofibroblast differentiation is currently largely unknown. PURPOSE: To determine the contribution of GSK-3 signalling in TGF-ß1-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-ß1 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-ß1-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-ß1-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 a functional antagonist of TGF-ß/smad signalling. CONCLUSION AND IMPLICATION: We demonstrate that GSK-3 signalling regulates TGF-ß1-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases.

The WNT signaling pathway from ligand secretion to gene transcription: molecular mechanisms and pharmacological targets.

Wingless/integrase-1 (WNT) signaling is a key pathway regulating various aspects of embryonic development; however it also underlies several pathological conditions in man, including various cancers and fibroproliferative diseases in several organs. Investigating the molecular processes involved in (canonical) WNT signaling will open new avenues for generating new therapeutics to specifically target diseases in which WNT signaling is aberrantly regulated. Here we describe the complexity of WNT signal transduction starting from the processes involved in WNT ligand biogenesis and secretion by WNT producing cells followed by a comprehensive overview of the molecular signaling events ultimately resulting in enhanced transcription of specific genes in WNT receiving cells. Finally, the possible targets for therapeutic intervention and the available pharmacological inhibitors for this complex signaling pathway are discussed.

Novel non-canonical TGF-ß signaling networks: emerging roles in airway smooth muscle phenotype and function.

The airway smooth muscle (ASM) plays an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD). ASM cells express a wide range of receptors involved in contraction, growth, matrix protein production and the secretion of cytokines and chemokines. Transforming growth factor beta (TGF-ß) is one of the major players in determining the structural and functional abnormalities of the ASM in asthma and COPD. It is increasingly evident that TGF-ß functions as a master switch, controlling a network of intracellular and autocrine signaling loops that effect ASM phenotype and function. In this review, the various elements that participate in non-canonical TGF-ß signaling, including MAPK, PI3K, WNT/ß-catenin, and Ca(2+), are discussed, focusing on their effect on ASM phenotype and function. In addition, new aspects of ASM biology and their possible association with non-canonical TGF-ß signaling will be discussed.

Noncanonical WNT-5A signaling regulates TGF-ß-induced extracellular matrix production by airway smooth muscle cells.

Transforming growth factor ß (TGF-ß), 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-ß-induced ECM expression by airway smooth muscle cells and demonstrate a novel link between TGF-ß 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-ß (4-16-fold; EC50 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-ß 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 (FZD8) and RYK attenuated TGF-ß-induced ECM expression. Collectively, these findings demonstrate that noncanonical WNT-5A signaling is activated by and necessary for TGF-ß-induced ECM production by airway smooth muscle cells, which could have significance in asthma pathogenesis.

Activation of WNT/ß-catenin signaling in pulmonary fibroblasts by TGF-ß1 is increased in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by abnormal extracellular matrix (ECM) turnover. Recently, activation of the WNT/ß-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 ß-catenin in fibroblast phenotype and function. METHODS: We assessed the expression of WNT-pathway genes and the functional role of ß-catenin, using MRC-5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. RESULTS: Pulmonary fibroblasts expressed mRNA of genes required for WNT signaling. Stimulation of fibroblasts with TGF-ß1, a growth factor important in COPD pathogenesis, induced WNT-5B, FZD8, DVL3 and ß-catenin mRNA expression. The induction of WNT-5B, FZD6, FZD8 and DVL3 mRNA by TGF-ß1 was higher in fibroblasts of individuals with COPD than without COPD, whilst basal expression was similar. Accordingly, TGF-ß1 activated ß-catenin signaling, as shown by an increase in transcriptionally active and total ß-catenin protein expression. Furthermore, TGF-ß1induced the expression of collagen1α1, α-sm-actin and fibronectin, which was attenuated by ß-catenin specific siRNA and by pharmacological inhibition of ß-catenin, whereas the TGF-ß1-induced expression of PAI-1 was not affected. The induction of transcriptionally active ß-catenin and subsequent fibronectin deposition induced by TGF-ß1 were enhanced in pulmonary fibroblasts from individuals with COPD. CONCLUSIONS: ß-catenin signaling contributes to ECM production by pulmonary fibroblasts and contributes to myofibroblasts differentiation. WNT/ß-catenin pathway expression and activation by TGF-ß1 is enhanced in pulmonary fibroblasts from individuals with COPD. This suggests an important role of the WNT/ß-catenin pathway in regulating fibroblast phenotype and function in COPD.