Tadashi Kohyama

Thyroid Transcription Factor-1 Inhibits Transforming Growth Factor-β–Mediated Epithelial-to-Mesenchymal Transition in Lung Adenocarcinoma Cells

Thyroid transcription factor-1 (TTF-1) is expressed in lung cancer, but its functional roles remain unexplored. TTF-1 gene amplification has been discovered in a part of lung adenocarcinomas, and its action as a lineage-specific oncogene is highlighted. Epithelial-to-mesenchymal transition (EMT) is a crucial event for cancer cells to acquire invasive and metastatic phenotypes and can be elicited by transforming growth factor-beta (TGF-beta). Mesenchymal-to-epithelial transition (MET) is the inverse process of EMT; however, signals that induce MET are largely unknown. Here, we report a novel functional aspect of TTF-1 that inhibits TGF-beta-mediated EMT and restores epithelial phenotype in lung adenocarcinoma cells. This effect was accompanied by down-regulation of TGF-beta target genes, including presumed regulators of EMT, such as Snail and Slug. Moreover, silencing of TTF-1 enhanced TGF-beta-mediated EMT. Thus, TTF-1 can exert a tumor-suppressive effect with abrogation of cellular response to TGF-beta and attenuated invasive capacity. We further revealed that TTF-1 down-regulates TGF-beta2 production in A549 cells and that TGF-beta conversely decreases endogenous TTF-1 expression, suggesting that enhancement of autocrine TGF-beta signaling accelerates the decrease of TTF-1 expression and vice versa. These findings delineate potential links between TTF-1 and TGF-beta signaling in lung cancer progression through regulation of EMT and MET and suggest that modulation of TTF-1 expression can be a novel therapeutic strategy for treatment of lung adenocarcinoma.

Erythromycin and clarithromycin attenuate cytokine-induced endothelin-1 expression in human bronchial epithelial cells

Erythromycin and its fourteen-member macrolide analogues have attracted attention for their efficacy in bronchial asthma. However, their mechanisms of action remain unclear. We evaluated the effects of the macrolide antibiotics on endothelin-1 (ET-1) expression in normal and transformed human bronchial epithelial cells, one of the sources of this potent bronchoconstrictor important in the pathogenesis of asthma. Human bronchial epithelial cells were obtained from the resected bronchi, and the effect of several antimicrobial and antiasthmatic drugs on the production and messenger ribonucleic acid (mRNA) levels of ET-1 was evaluated. Bronchoepithelial cells were also isolated from the mucosa of asthmatic patients under fibreoptic bronchoscopy, and the modulating effects of the drugs were studied. Erythromycin and clarithromycin uniquely suppressed mRNA levels as well as the release of ET- at therapeutic and non-cytotoxic concentrations (percentage inhibition of ET-1 protein release: 26.4+/-5.22% and 31.2+/-7.45%, respectively, at 10(-6) M). Furthermore, erythromycin and clarithromycin inhibited ET-1 expression in bronchoepithelial cells from patients with chronic, stable asthma. A glucocorticosteroid, dexamethasone, also inhibited ET-1 expression. In contrast, theophylline, salbutamol and FK506 had no effect on ET-1 production. Our findings demonstrated that these fourteen-member macrolide antibiotics had an inhibitory effect on endothelin-1 expression in human bronchial epithelial cells. Moreover, this new mode of action may have some relevance to their clinical efficacy in bronchial asthma.

Tumor necrosis factor-α enhances both epithelial-mesenchymal transition and cell contraction induced in A549 human alveolar epithelial cells by transforming growth factor-β1

ABSTRACT Recently, epithelial-mesenchymal transition (EMT) has been reported to contribute to tissue fibrosis through enhanced transforming growth factor (TGF)-β1 signaling. Tumor necrosis factor (TNF)-α has also been implicated in tissue fibrosis. Therefore, the authors investigated whether TNF-α affected TGF-β1–induced EMT. Cultured alveolar epithelial cells (A549 cells) were stimulated with TGF-β1 (5 ng/mL), with/without TNF-α (10 ng/mL). TGF-β1 induced EMT of A549 cells, with loss of E-cadherin and acquisition of vimentin. Combination of TNF-α with TGF-β1 enhanced EMT, causing morphological changes, while quantitative polymerase chain reaction (PCR) showed suppression of E-cadherin mRNA and expression of vimentin mRNA. In addition, the gel contraction method revealed that cells that had undergone EMT acquired cell contractility, which is a feature of mesenchymal cells. Stimulation with TGF-β1 induced cell contraction, as did TNF-α. Moreover, costimulation with TGF-β1 and TNF-α enhanced the cell contraction. Although IFN-γ suppressed spontaneous cell contraction, it did not suppress cell contraction, which was induced by TGF-β1. In conclusion, TNF-α enhances not only EMT but also cell contraction induced by TGF-β1. EMT might contribute to tissue fibrosis through induction of cell contraction.

Matrix metalloproteinase-9 activates TGF-β and stimulates fibroblast contraction of collagen gels

Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in three-dimensional (3D) collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 small-interfering RNA (siRNA) were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without transforming growth factor (TGF)-β1 for 5 days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA-treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a nonspecific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9-deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9-deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9-deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-β1 and reduced several TGF-β1-driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Because TGF-β1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-β1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3D collagen gels mediated by fibroblasts.

Characterization of human lung cancer-associated fibroblasts in three-dimensional in vitro co-culture model.

Lung cancer is the most common cause of cancer-related death worldwide. Stromal cancer-associated fibroblasts (CAFs) play crucial roles in carcinogenesis, proliferation, invasion, and metastasis of non-small cell lung carcinoma, and targeting of CAFs could be a novel strategy for cancer treatment. However, the characteristics of human CAFs still remain to be better defined. In this study, we established patient-matched CAFs and normal fibroblasts (NFs), from tumoral and non-tumoral portions of resected lung tissue from lung cancer patients. CAFs showed higher α-smooth muscle actin (α-SMA) expression than NFs, and CAFs clearly enhanced collagen gel contraction. Furthermore, we employed three-dimensional co-culture assay with A549 lung cancer cells, where CAFs were more potent in inducing collagen gel contraction. Hematoxylin and eosin staining of co-cultured collagen gel revealed that CAFs had the potential to increase invasion of A549 cells compared to NFs. These observations provide evidence that lung CAFs have the tumor-promoting capacity distinct from NFs.

Simultaneous Stimulation with TGF-β1 and TNF-α Induces Epithelial Mesenchymal Transition in Bronchial Epithelial Cells

Background: Airway remodeling is an important feature of chronic airway disease, but the mechanisms involved remain unclear. Recently, epithelial mesenchymal transition (EMT) was reported to be associated with tissue fibrosis. TGF-β1, which is a potent inducer of EMT, is thought to be related to the pathogenesis of airway remodeling. We investigated whether TGF-β1 and/or TNF-α induce EMT in bronchial epithelial cells. Methods: Cultured BEAS-2B cells and primary normal human bronchial epithelial cells (NHBE) were treated with TGF-β1 and/or TNF-α. Morphological changes and the expression of EMT-related markers were evaluated by immunocytochemical staining. Expressions of EMT-related markers, extracellular matrix (ECM) components (collagen type I and versican), and TGF-β receptors I, II, and III were analyzed by quantitative RT-PCR. Migration was evaluated using the Boyden chamber technique. Results: The TGF-β1-induced EMT in BEAS-2B cells was demonstrated on the basis of morphological changes and the downregulation of E-cadherin. Costimulation with TNF-α enhanced the TGF-β1-induced morphological changes and increased vimentin expression. Treatment with TGF-β1 increased the expression of collagen type I and versican. EMT induced with TGF-β1 plus TNF-α promoted cell migration. Stimulation of NHBE with TGF-β1 led to EMT. Conclusion: TGF-β1 induced EMT in BEAS-2B cells, and costimulation with TNF-α enhanced the EMT. As a result of the EMT process, BEAS-2B cells acquired functions of mesenchymal cells. In addition, TGF-β1 treatment induced EMT in NHBE as shown by changes in EMT-related markers. Bronchial epithelial cells might contribute to airway remodeling through EMT.

Cigarette smoke extract inhibits chemotaxis and collagen gel contraction mediated by human bone marrow osteoprogenitor cells and osteoblast-like cells

Cell migration and matrix remodeling are key events in tissue repair and restructuring. Osteoblasts are responsible for the production of new bone matrix during bone remodeling. The activity of these cells can be modulated by a number of factors. The current study evaluated the hypothesis that cigarette smoke extract can alter repair and remodeling responses of human osteoprogenitor cells and osteoblast-like cells and, therefore, could explain one mechanism by which cigarette smoking leads to osteoporosis. Human osteoprogenitor cells were isolated from normal human bone marrow and maintained in culture under either control conditions or conditions that induced differentiation into osteoblast-like cells. Both cell types migrated toward fibronectin and PDGF-BB as chemoattractants. Neither responded to TGF-β1. The osteoprogenitor cells were more active in their chemotactic response. The chemotactic response of both cell types was inhibited by cigarette smoke extract in a concentration-dependent manner. Both cell types, when cultured in three-dimensional native collagen gels maintained in floating culture, induced contraction of their surrounding matrices. Contraction was augmented by serum, PDGF-BB, and TGF-β1. Osteoprogenitor cells were less active in inducing contraction than were osteoblast-like cells. Contraction of both cell types was inhibited by cigarette smoke extract. Cigarette smoke extract also inhibited the production of fibronectin by both cell types maintained in three-dimensional culture. Addition of exogenous fibronectin partially restored the ability of the cells to contract three-dimensional collagen gels. The current study demonstrates that cigarette smoke can interfere with the ability of bone cells to participate in repair and remodeling events. Such an effect may be one mechanism leading to the development of osteoporosis.

Glucocorticoids Modulate TGF-β Production by Human Fetal Lung Fibroblasts

TGF-β is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF-β isoforms affect TGF-β production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-β1, -β2, or -β3. Post-culture media were collected for ELISA assays of TGF-β1, -β2, and -β3 . TGF-β mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-β2 and -β3 stimulated TGF-β1 production significantly (p < 0.01 relative to control). TGF-β1 stimulated TGF-β2 production (p < 0.01 relative to control). TGF-β3 was undetectable. Glucocorticoids significantly inhibited TGF-β1 and TGF-β2 production and reduced expression of the up-regulated TGF-β1 and TGF-β2 mRNA induced by exogenous TGF-β1, -β2, or -β3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF-β-stimulated cells, it was reduced by glucocorticoids. Thus, TGF-β isoforms may stimulate production of various TGF-β isoforms in the lung. Glucocorticoids then may block TGF-β production by modulating mRNA levels and c-Jun.

Cigarette Smoke Stimulates MMP‐1 Production by Human Lung Fibroblasts Through the ERK1/2 Pathway

An imbalance between proteases and anti‐proteases is believed to play an important role in the pathogenesis of emphysema. In this study, we explored the hypothesis that cigarette smoke can alter tissue structure through an effect on the release of matrix metalloproteinase‐1 (MMP‐1) and type I tissue inhibitor of metalloproteinases (TIMP‐1). Cigarette smoke extract (CSE) significantly stimulated pro‐MMP‐1 production (determined by ELISA and immunoblots) and mRNA expression (by real‐time RT‐PCR) by human fetal lung fibroblasts (HFL‐1) in a concentration‐dependent manner (2.5–10%). High concentrations of CSE (10%) could potentially activate the latent form of MMP‐1 as the high molecular weight (52 kDa) form was converted into a low molecular weight (42 kDa) form consistent with active MMP‐1. TIMP‐1 production, however, was not significantly altered by the concentrations of CSE tested. After 30 min exposure, CSE significantly induced ERK1/2 phosphorylation, which then gradually decreased from 90 minutes to 3 hours. PD98059, a specific inhibitor of ERK‐MAPK, significantly blocked the CSE effect on ERK1/2 phosphorylation. Furthermore, PD98059 significantly inhibited the CSE effect on MMP‐1 production and mRNA expression by fibroblasts. These results suggest that cigarette smoke stimulates production and likely activates MMP‐1 through activating ERK1/2 signal transduction pathway. By inducing MMP‐1, cigarette smoke may result in excess tissue destruction and contribute to the development of emphysema.

Glucocorticoids and TGF-β1 Synergize in Augmenting Fibroblast Mediated Contraction of Collagen Gels

TGF-β plays a central role in the initiation and progression of pulmonary fibrosis. Glucocorticoids are frequently used to treat fibrotic diseases, but beneficial effects are often modest. Both TGF-β and glucocorticoids have been reported to increase fibroblast contraction of native collagen gels, a model of fibrotic tissue remodeling. Therefore, we sought to determine how glucocorticoids interact with TGF-β in this system. In this study, human fetal lung fibroblasts (HFL-1) were pretreated with or without TGF-β for 72 h before they were cast into type I collagen gels. Various concentrations of glucocorticoids (budesonide or hydrocortisone) were added at the time of casting. Gel size was then monitored at different times after gel release. The surrounding media were collected for the assay of prostaglandin E2 (PGE2) and the cell lysates were analyzed for cyclooxygenase (COX) expression by immunoblot. Glucocorticoids alone significantly enhanced fibroblast-mediated contraction of collagen gels (P < 0.01) and dose-dependently inhibited PGE2 release by HFL-1 fibroblasts. TGF-β significantly augmented gel contraction but also induced a 30% increase in PGE2 release and increased the expression of COX-1. Glucocorticoids inhibited TGF-β1 induced-PGE2 release, and enhanced TGF-β augmented gel contraction without significantly affecting TGF-β augmented COX-1 expression. Indomethacin, a COX inhibitor, increased TGF-β augmented gel contraction but had no further effect when added together with glucocorticoids. Thus, glucocorticoids can synergize with TGF-β in augmenting fibroblast mediated collagen gel contraction through the inhibition of PGE2 production. Such interactions between glucocorticoids and TGF-β may account, in part, for the lack of response of fibrotic diseases to glucocorticoids.