Thrombin induces epithelial-mesenchymal transition via PAR-1, PKC, and ERK1/2 pathways in A549 cells.

Thrombin activates protease-activated receptor (PAR)-1 and induces a myofibroblast phenotype in normal lung fibroblasts. The origins of myofibroblasts are resident fibroblasts, fibrocytes, and epithelial-mesenchymal transition (EMT). We investigated the effects of thrombin, an important mediator of interstitial lung fibrosis, on EMT in A549 human alveolar epithelial cells. We show that thrombin induced EMT and collagen I secretion through the activation of PAR-1, and PKC and ERK1/2 phosphorylation in A549 cells. These effects were largely prevented by a specific PAR-1 antagonist, short interfering RNA (siRNA) directed against PAR-1, or specific PKCα/ß, δ, and ε inhibitors. These data indicated that interaction with thrombin and alveolar epithelial cells might directly contribute to the pathogenesis of pulmonary fibrosis through EMT. Targeting PAR-1 on the pulmonary epithelium or specific inhibitors to PKCα/ß, δ, and ε might stop the fibrotic processes in human idiopathic pulmonary fibrosis by preventing thrombin-induced EMT.

Effects of elastase inhibitor on the epithelial cell apoptosis in bleomycin-induced pulmonary fibrosis.

Alveolar epithelial cell injury and apoptosis is consistent findings in human idiopathic pulmonary fibrosis (IPF). Epithelial cell apoptosis is known to be induced by leukocyte elastase in vitro. The authors hypothesized that synthetic neutrophil elastase inhibitor, sivelestat (ONO-5046), can inhibit the bleomycin-induced pulmonary fibrosis in rats by blocking the apoptotic pathways in epithelial cells. Adult rats were injected with intratracheal bleomycin. Sivelestat was given for 13 days intraperitoneally after bleomycin treatments. Similar experiments were carried out in which A549 cells, a human alveolar type II epithelial cell line, were treated with bleomycin or neutrophil elastase. In rats, sivelestat decreased neutrophil counts and the cytokine-induced neutrophil chemoattractant (CINC)-1 in the bronchoalveolar lavage (BAL) fluid of bleomycin-treated rats. Sivelestat also decreased the bleomycin-induced lung inflammatory cell apoptosis by decreasing caspase-3 and -9 activities. In A549 cells, sivelestat decreased the elastase-induced epithelial cell apoptosis but not the bleomycin-induced epithelial cell apoptosis. Similarly, sivelestat inhibited the elastase-induced cell death but not the bleomycin-induced cell death in MTT assays. Sivelestat also inhibited the elastase-induced caspase-3 and -9 activities and cytochrome c release from the mitochondria but did not inhibit the bleomycin-induced caspase activities in A549 cells. In conclusion, bleomycin caused the lung inflammatory cell apoptosis through the caspase-9 and -3 pathways in rats. Sivelestat inhibited pulmonary fibrosis by blocking these mitochondria-mediated apoptotic pathways in bleomycin-treated rats and in elastase-treated A549 cells. These findings suggest that sivelestat can suppress the bleomycin-induced pulmonary fibrosis by blocking neutrophil chemotaxis and by inhibiting the neutrophil elastase-induced lung cell apoptosis in rats.

Nitric oxide induces MUC5AC mucin in respiratory epithelial cells through PKC and ERK dependent pathways.

BACKGROUND: Nitric oxide (NO) is generally increased during inflammatory airway diseases. This increased NO stimulates the secretion of mucin from the goblet cell and submucosal glands but the mechanism is still unknown precisely. In this study, we investigated potential signaling pathways involving protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the NO-induced MUC5AC mucin gene and protein expression in A549 cells. METHODS: Nitric oxide was donated to the A549 cells by NOR-1. MUC5AC mucin levels were assayed by enzyme-linked immunosorbent assay (ELISA). MUC5AC promoter activity was determined by measuring luciferase activity after the lysing the transfected cells. Activation of PKC isoforms were measured by assessing the distribution of the enzyme between cytosolic and membrane fractions using immunoblotting. Immunoblotting experiments using a monoclonal antibody specific to PKC isoforms were performed in the cytosol and membrane fractions from A549 cells. Western blot analysis for pERK and p38 were performed using the corresponding antibodies from the cell lysates after donating NO to the A549 cells by NOR-1. RESULTS: The transcriptional activity of MUC5AC promoter was maximal at the concentration of 0.1 mM NOR-1 for 1 hour incubation in transfected A549 cells. (+/-)-(E)-methyl-2-((E)-hydroxyimino)-5-nitro-6-methoxy-3-hexenamide (NOR-1) markedly displaced the protein kinase C (PKC)alpha and PKCdelta from the cytosol to the membrane. Furthermore, the PKC-alpha,betainhibitors, GO6976 (10 nM) and PKCdelta inhibitors, rottlerin (4 muM) inhibited the NOR-1 induced migration of PKCalpha and PKCdelta respectively. NOR-1 also markedly increased the MUC5AC promoter activity and mRNA expression, mucin synthesis and ERK1/2 phosphorylation. The PKC inhibitors also inhibited the NOR-1 induced MUC5AC mRNA and MUC5AC protein synthesis by inhibiting the activation of PKCalpha and PKCdelta with ERK1/2 pathways. CONCLUSION: Exogenous NO induced the MUC5AC mucin gene and protein through the PKCalpha and PKCdelta-ERK pathways in A549 cells. Inhibition of PKC attenuated NO-mediated MUC5AC mucin synthesis. In view of this findings, PKC inhibitors might be useful in the treatment of bronchial asthma and chronic bronchitis patients where NO and mucus are increased in the bronchial airways.

Titanium dioxide particle-induced goblet cell hyperplasia: association with mast cells and IL-13.

BACKGROUND: Inhalation of particles aggravates respiratory symptoms including mucus hypersecretion in patients with chronic airway disease and induces goblet cell hyperplasia (GCH) in experimental animal models. However, the underlying mechanisms remain poorly understood. METHODS: To understand this, the numbers of goblet cells, Muc5ac (+) expressing epithelial cells and IL-13 expressing mast cells were measured in the trachea of sham or TiO2 particles-treated rats using periodic acid-Schiff, toluidine blue and immunohistochemical staining. RT-PCR for Muc-1, 2 and 5ac gene transcripts was done using RNA extracted from the trachea. Differential cell count and IL-13 levels were measured in bronchoalveolar lavage (BAL) fluid. In pretreatment groups, cyclophosphamide (CPA) or dexamethasone (DEX) was given before instillation of TiO2. TiO2 treatment markedly increased Muc5ac mRNA expression, and Muc5ac (+) or PAS (+) epithelial cells 48 h following treatment. RESULTS: The concentration of IL-13 in BAL fluids was higher in TiO2 treated-rats when compared to those in sham rats (p < 0.05). Pretreatment with cyclophosphamide (CPA) decreased the number of neutrophils and eosinophils in BAL fluid of TiO2 treated-rats (p < 0.05), but affected neither the percentage of PAS (+) cells, nor IL-13 levels in the BAL fluids (p > 0.05). In contrast, pretreatment with dexamethasone (DEX) diminished the percentage of PAS (+) cells and the levels of IL-13 (p < 0.05). TiO2 treatment increased the IL-13 (+) mast cells (p < 0.05) in the trachea, which was suppressed by DEX (p < 0.05), but not by CPA pretreatment (p > 0.05). In addition there were significant correlations of IL-13 (+) rate of mast cells in the trachea with IL-13 concentration in BAL fluid (p < 0.01) and with the percentage of Muc5ac (+) cells in the sham and TiO2 treated rats (p < 0.05). CONCLUSION: In conclusion, TiO2 instillation induces GCH and Muc5ac expression, and this process may be associated with increased production of IL-13 by mast cells.

Inhibitory effect of receptor for advanced glycation end products (RAGE) on the TGF-ß-induced alveolar epithelial to mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is a lethal parenchymal lung disease characterized by myofibroblast proliferation. Alveolar epithelial cells (AECs) are thought to produce myofibroblasts through the epithelial to mesenchymal transition (EMT). Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation is associated with renal fibrosis during diabetes and liver fibrosis. RAGE is expressed at low basal levels in most adult tissues except the lung. In this study, we evaluated the interaction of ligand advanced glycation end products (AGE) with RAGE during the epithelial to myofibroblast transition in rat AECs. Our results indicate that AGE inhibited the TGF-ß-dependent alveolar EMT by increasing Smad7 expression, and that the effect was abolished by RAGE siRNA treatment. Thus, the induction of Smad7 by the AGE-RAGE interaction limits the development of pulmonary fibrosis by inhibiting TGF-ß-dependent signaling in AECs.

Inhibitory effect of CXC chemokine receptor 4 antagonist AMD3100 on bleomycin induced murine pulmonary fibrosis.

CXC chemokine receptor 4 (CXCR4), which binds the stromal cell-derived factor-1 (SDF-1), has been shown to play a critical role in mobilizing the bone marrow (BM)-derived stem cells and inflammatory cells. We studied the effects of AMD3100, CXCR4 antagonist, on a murine bleomycin-induced pulmonary fibrosis model. Treatment of mice with AMD3100 in bleomycin-treated mice resulted in the decrease of SDF-1 in bronchoalveolar lavage (BAL) fluids at an early stage and was followed by the decrease of fibrocytes in the lung. AMD3100 treatment decreased the SDF-1 mRNA expression, fibrocyte numbers in the lung at an early stage (day 3) and CXCR4 expression at the later stage (day 7 and 21) after bleomycin injury. The collagen content and pulmonary fibrosis were significantly attenuated by AMD3100 treatment in later stage of bleomycin injury. AMD3100 treatment also decreased the murine mesenchymal and hematopoietic stem cell chemotaxis when either in the stimulation with bleomycin treated lung lysates or SDF-1 in vitro. In BM stem cell experiments, the phosphorylation of p38 MAPK which was induced by SDF-1 was significantly blocked by addition of AMD3100. Our data suggest that AMD3100 might be effective in preventing the pulmonary fibrosis by inhibiting the fibrocyte mobilization to the injured lung via blocking the SDF-1/CXCR4 axis.

Interleukin-25 and interleukin-13 production by alveolar macrophages in response to particles.

Particle inhalation-induced lung inflammation acts as an adjuvant to allergens or respiratory viral infection in a process that is mediated by macrophages and epitheliums. The production of interleukin (IL)-4 and IL-13 by activated T cells is involved in the augmentation of Th2-type immune responses to particles, and IL-25 induces the synthesis of IL-4 and IL-13. However, whether IL-13 and IL-25 are directly regulated by particle instillation in the lung has not been studied. The aim of this study was to reveal particle induction of IL-13 and IL-25 in the lung. TiO(2) instillation potently induced the mRNA expression for IL-25 and IL-13 in lung tissue extracts 24 h after treatment, as compared with the sham group. Immunostaining for IL-25 and IL-13 showed strong positivity for macrophages in the inflammatory lung lesions of TiO(2)-treated rats. The alveolar macrophages expressed IL-25 and IL-13 24 h after in vitro stimulation with TiO(2) particles in dose- and time-dependent manners, with maximal induction at 24 and 48 h after stimulation, respectively. The sequence of the rat IL-25 gene is 95% homologous with the mouse IL-25 gene. These findings indicate that alveolar macrophages play an important role in particle-induced lung inflammation via direct induction of IL-13 and IL-25 production.