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-β1 (TGF-β1) release and signaling. Furthermore, CS exposure reduced the expression of E-cadherin, which was prevented by applying a TGF-β1 neutralizing antibody. Exposure of BEAS-2B cells cultured in 2D to diesel exhaust particles (DEP) increased TGF-β1 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-β 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

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

Activation of WNT/beta-Catenin Signaling in Pulmonary Fibroblasts by TGF-beta(1) Is Increased in Chronic Obstructive Pulmonary Disease

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

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

<p>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. (C) 2013 Elsevier Inc. All rights reserved.</p>

beta-Catenin regulates airway smooth muscle contraction

Jansen SR, Van Ziel AM, Baarsma HA, Gosens R. beta-Catenin regulates airway smooth muscle contraction. Am J Physiol Lung Cell Mol Physiol 299: L204-L214, 2010. First published May 14, 2010; doi:10.1152/ajplung.00020.2010.-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

beta-Catenin signaling is required for TGF-beta(1)-induced extracellular matrix production by airway smooth muscle cells

Baarsma HA, Menzen MH, Halayko AJ, Meurs H, Kerstjens HA, Gosens R. beta-Catenin signaling is required for TGF-beta(1)-induced extracellular matrix production by airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 301: L956-L965, 2011. First published September 9, 2011; doi: 10.1152/ajplung.00123.2011.-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-beta(1) (TGF-beta(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 beta-catenin signaling and its contribution to ECM production by airway smooth muscle cells in response to TGF-beta(1). Stimulation of airway smooth muscle cells with TGF-beta(1) resulted in a time-dependent increase of total and nonphosphorylated beta-catenin protein expression via induction of beta-catenin mRNA and inhibition of GSK-3. In addition, the TGF-beta(1)-induced beta-catenin activated TCF/LEF-dependent gene transcription, as determined by the beta-catenin sensitive TOP-flash luciferase reporter assay. Furthermore, TGF-beta(1) stimulation increased mRNA expression of collagen I alpha 1, fibronectin, versican, and PAI-1. Pharmacological inhibition of beta-catenin by PKF115-584 or down-regulation of beta-catenin expression by specific small interfering RNA (siRNA) substantially inhibited TGF-beta(1)-induced expression of the ECM genes. Fibronectin protein deposition by airway smooth muscle cells in response to TGF-beta(1) was also inhibited by PKF115-584 and beta-catenin siRNA. Moreover, transfection of airway smooth muscle cells with a nondegradable beta-catenin mutant (S33Y beta-catenin) was sufficient for inducing fibronectin protein expression. Collectively, these findings indicate that beta-catenin signaling is activated in response to TGF-beta(1) in airway smooth muscle cells, which is required and sufficient for the regulation of ECM protein production. Targeting beta-catenin-dependent gene transcription may therefore hold promise as a therapeutic intervention in airway remodeling in both asthma and COPD

beta-Catenin signaling is required for TGF-beta(1)-induced extracellular matrix production by airway smooth muscle cells

Baarsma HA, Menzen MH, Halayko AJ, Meurs H, Kerstjens HA, Gosens R. beta-Catenin signaling is required for TGF-beta(1)-induced extracellular matrix production by airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 301: L956-L965, 2011. First published September 9, 2011; doi: 10.1152/ajplung.00123.2011.-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-beta(1) (TGF-beta(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 beta-catenin signaling and its contribution to ECM production by airway smooth muscle cells in response to TGF-beta(1). Stimulation of airway smooth muscle cells with TGF-beta(1) resulted in a time-dependent increase of total and nonphosphorylated beta-catenin protein expression via induction of beta-catenin mRNA and inhibition of GSK-3. In addition, the TGF-beta(1)-induced beta-catenin activated TCF/LEF-dependent gene transcription, as determined by the beta-catenin sensitive TOP-flash luciferase reporter assay. Furthermore, TGF-beta(1) stimulation increased mRNA expression of collagen I alpha 1, fibronectin, versican, and PAI-1. Pharmacological inhibition of beta-catenin by PKF115-584 or down-regulation of beta-catenin expression by specific small interfering RNA (siRNA) substantially inhibited TGF-beta(1)-induced expression of the ECM genes. Fibronectin protein deposition by airway smooth muscle cells in response to TGF-beta(1) was also inhibited by PKF115-584 and beta-catenin siRNA. Moreover, transfection of airway smooth muscle cells with a nondegradable beta-catenin mutant (S33Y beta-catenin) was sufficient for inducing fibronectin protein expression. Collectively, these findings indicate that beta-catenin signaling is activated in response to TGF-beta(1) in airway smooth muscle cells, which is required and sufficient for the regulation of ECM protein production. Targeting beta-catenin-dependent gene transcription may therefore hold promise as a therapeutic intervention in airway remodeling in both asthma and COPD

De novo synthesis of beta-catenin via H-Ras and MEK regulates airway smooth muscle growth

beta-Catenin is a component of adherens junctions that also acts as a transcriptional coactivator when expressed in the nucleus. Growth factors are believed to regulate the nuclear expression of beta-catenin via inactivation of glycogen synthase kinase 3 (GSK-3) by phosphorylation, resulting in increased beta-catenin protein stability. Here, we report on a novel pathway that regulates the expression and nuclear presence of beta-catenin. In proliferating human airway smooth muscle cells, we observed increased expression of beta-catenin, which was required for proliferation. Interestingly, increased beta-catenin expression was accompanied by an increase in beta-catenin mRNA and was independent of beta-catenin liberation from the plasma membrane, suggesting a role for de novo synthesis. This was confirmed using actinomycin D and cycloheximide, which abrogated the induction and nuclear localization of beta-catenin protein. GSK-3 inhibition using SB216763 failed to regulate beta-catenin mRNA. However, expression of dominant negative H-Ras or pharmacological inhibition of MEK reduced serum and TGF-beta-induced beta-catenin mRNA and protein. Collectively, these data indicate that beta-catenin is an important signaling intermediate in airway smooth muscle growth and that its cellular accumulation and nuclear localization require de novo protein synthesis effected, in part, via H-Ras and MEK.-Gosens, R., Baarsma, H. A., Heijink, I. H., Oenema, T. A., Halayko, A. J., Meurs, H., Schmidt, M. De novo synthesis of beta-catenin via H-Ras and MEK regulates airway smooth muscle growth. FASEB J. 24, 757-768 (2010). www.fasebj.or

Novel non-canonical TGF-beta signaling networks:Emerging roles in airway smooth muscle phenotype and function

<p>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-beta) 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-beta 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-beta signaling, including MAPK, PI3K, WNT/beta-catenin, and Ca2+, 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-beta signaling will be discussed. (C) 2012 Elsevier Ltd. All rights reserved.</p>