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Kana Aoyagi-Ikeda,
Toshitaka Maeno,
Hiroki Matsui,
Manabu Ueno, Kenichiro Hara,
Yasuhiro Aoki,
Fumiaki Aoki,
Takehisa Shimizu,
Hiroshi Doi,
Keiko Kawai-Kowase,
Tatsuya Iso,
Tatsuo Suga,
Masashi Arai,
Masahiko Kurabayashi
[show abstract]
[hide abstract]
ABSTRACT: Notch is an ancient cell-signaling system that regulates the specification of cell fate. This study examined the role of Notch in the epithelial-mesenchymal transition (EMT) and myofibroblast differentiation of cultured RLE-6TN cells (i.e., rat alveolar epithelial cells). The activation of Notch, either by ectopic expression of the Notch intracellular domain or by the co-culture of RLE-6TN cells with L-Jagged1 cells, induces the expression of smooth muscle α-actin (SMA) and other mesenchymal marker genes (collagen I and vimentin), and reduces the expression of epithelial marker genes (E-cadherin, occludin, and zonula occludens-1). The pharmacologic inhibition of the endogenous Notch signal significantly inhibited the transforming growth factor-β (TGF-β)-induced expression of SMA. Cell migratory capacity was increased by Notch. Luciferase assays revealed that the CC(A/T)(6)GG (CArG) box and the TGF-β control element (TCE) are required for Notch-induced SMA gene transcription. DNA microarray analysis revealed that members of the TGF-β family as well as Jagged1 were induced in RLE-6TN cells by Notch. Western blot analysis showed that Notch induced the phosphorylation of Smad3, and the TGF-β receptor type I/activin receptor-like kinase 5 (ALK5) kinase inhibitor SB431542 markedly reduced the Notch-induced expression of SMA. Enzyme-linked immunosorbent assays confirmed the production of TGF-β1 from RLE-6TN cells by Notch. Immunohistochemistry of a bleomycin-induced model of pulmonary fibrosis and lung specimens from patients with idiopathic interstitial pneumonias showed that Notch was strongly expressed in myofibroblasts, identified as SMA-positive cells. These data indicate that Notch induces myofibroblast differentiation through a TGF-β-Smad3 pathway that activates SMA gene transcription in a CArG-dependent and TCE-dependent manner in alveolar epithelial cells. Our data also imply that Notch induces the EMT phenotype, with increased migratory behavior in pulmonary fibrosis.
American Journal of Respiratory Cell and Molecular Biology 07/2011; 45(1):136-44. · 5.13 Impact Factor
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[show abstract]
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ABSTRACT: Hypoxia-inducible factor-1α (HIF-1α), a transcription factor that functions as a master regulator of oxygen homeostasis, has been implicated in fibrinogenesis. Here, we explore the role of HIF-1α in transforming growth factor-β (TGF-β) signaling by examining the effects of TGF-β(1) on the expression of plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of lung tissue from a mouse bleomycin (BLM)-induced pulmonary fibrosis model revealed that expression of HIF-1α and PAI-1 was predominantly induced in alveolar macrophages. Real-time RT-PCR and ELISA analysis showed that PAI-1 mRNA and activated PAI-1 protein level were strongly induced 7 days after BLM instillation. Stimulation of cultured mouse alveolar macrophages (MH-S cells) with TGF-β(1) induced PAI-1 production, which was associated with HIF-1α protein accumulation. This accumulation of HIF-1α protein was inhibited by SB431542 (type I TGF-β receptor/ALK receptor inhibitor) but not by PD98059 (MEK1 inhibitor) and SB203580 (p38 MAP kinase inhibitor). Expression of prolyl-hydroxylase domain (PHD)-2, which is essential for HIF-1α degradation, was inhibited by TGF-β(1), and this decrease was abolished by SB431542. TGF-β(1) induction of PAI-1 mRNA and its protein expression were significantly attenuated by HIF-1α silencing. Transcriptome analysis by cDNA microarray of MH-S cells after HIF-1α silencing uncovered several pro-fibrotic genes whose regulation by TGF-β(1) required HIF-1α, including platelet-derived growth factor-A. Taken together, these findings expand our concept of the role of HIF-1α in pulmonary fibrosis in mediating the effects of TGF-β(1) on the expression of the pro-fibrotic genes in activated alveolar macrophages.
AJP Lung Cellular and Molecular Physiology 01/2011; 300(5):L740-52. · 3.66 Impact Factor
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Kana Aoyagi-Ikeda,
Toshitaka Maeno,
Hiroki Matsui,
Manabu Ueno, Kenichiro Hara,
Yasuhiro Aoki,
Fumiaki Aoki,
Takehisa Shimizu,
Hiroshi Doi,
Keiko Kawai-Kowase,
Tatsuya Iso,
Tatsuo Suga,
Masashi Arai,
Masahiko Kurabayashi
[show abstract]
[hide abstract]
ABSTRACT: Notch is an ancient cell signaling system that regulates cell fate specification. This study examined the role of Notch in epithelial-mesenchymal transition (EMT) and myofibroblast differentiation of cultured RLE-6TN cells, rat alveolar epithelial cells. Activation of Notch, either by ectopic expression of Notch intracellular domain or by coculture of RLE-6TN cells with L-Jagged1 cells, induced the expression of the smooth muscle α-actin (SMA) and other mesenchymal marker genes (collagen I and vimentin) and reduced the expression of the epithelial marker genes (E-cadherin, occludin and zonula occludens-1). Pharmacological inhibition of endogenous Notch signal significantly inhibited transforming growth factor-β (TGF-β)-induced SMA expression. Cell migratory capacity was increased by Notch. Luciferase assays revealed that CArG box and TGF-β-control element (TCE) are required for Notch-induced SMA gene transcription. DNA microarray analysis revealed that members of TGF-β family as well as Jagged1 were induced in RLE-6TN cells by Notch. Western blot analysis showed that Notch induced phosphorylation of Smad3 and TGF-β receptor typeI/ALK5 kinase inhibitor SB431542 markedly reduced Notch-induced SMA expression. Enzyme-linked immunosorbent assays confirmed the production of TGF-β1 from RLE-6TN cells by Notch. Immunohistochemistry of bleomycin-induced pulmonary fibrosis model and lung specimens from idiopathic interstitial pneumonias patients showed that Notch was strongly expressed in myofibroblasts as identified as SMA-positive cells. These data indicate that Notch induces myofibroblast differentiation through TGF-ß/Smad3 pathway which activates SMA gene transcription in a CArG- and TCE-dependent manner in alveolar epithelial cells. Our data also imply that Notch induces EMT phenotype with an increased migratory behavior in pulmonary fibrosis.
American Journal of Respiratory Cell and Molecular Biology 09/2010; · 5.13 Impact Factor
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Kyoichi Kaira,
Noriaki Sunaga,
Noriko Yanagitani,
Tadayoshi Kawata,
Mitsuyoshi Utsugi,
Kimihiro Shimizu,
Takeshi Ebara,
Hidemasa Kawamura,
Tetsuo Nonaka,
Hitoshi Ishikawa,
Hideyuki Sakurai,
Tatsuo Suga, Kenichiro Hara,
Takeshi Hisada,
Tamotsu Ishizuka,
Takashi Nakano,
Masatomo Mori
[show abstract]
[hide abstract]
ABSTRACT: To determine the maximum tolerated dose (MTD) and recommended dose (RD) of S-1 in combination with cisplatin and thoracic radiotherapy in patients with unresectable Stage III non-small-cell lung cancer (NSCLC).
S-1 was administered orally twice daily for 14 days and cisplatin on Days 1 and 8 of each cycle; this was repeated every 3 weeks. Doses of each drug were planned as follows: level 0, 50/40; level 1, 60/40; level 2, 70/40; level 3, 80/40 (S-1 [mg/m(-2)/day(-1)]/cisplatin [mg/m(-2)/day(-1)]). Thoracic radiation therapy was administered in 2 Gy fractions five times weekly to a total dose of 60 Gy.
Ten patients were enrolled in this study. All patients received 60 Gy of thoracic radiotherapy and 7 (70%) patients received four cycles of chemotherapy. At level 1, 2 of 3 patients experienced a delay exceeding 10 days in the cisplatin administration of Day 29. Grade 4 neutropenia and Grade 3 fever occurred in 1 and 1 patients, respectively. Nonhematologic toxicities were mild. None developed >or=Grade 3 esophagitis or lung toxicity. At level 0, 2 of 7 patients developed dose-limiting toxicity. Thus, level 1 was considered the MTD and Level 0 was selected as the RD. Objective responses were seen in all patients.
The RD is the level 0 dose, and this regimen is a feasible and well-tolerated regimen for the treatment of patients with Stage III NSCLC.
International journal of radiation oncology, biology, physics 10/2008; 75(1):109-14. · 4.59 Impact Factor
