Notch induces myofibroblast differentiation of alveolar epithelial cells via transforming growth factor-{beta}-Smad3 pathway

• Published in: American journal of respiratory cell and molecular biology Vol. 45; no. 1; pp. 136 - 144
• Main Authors: Aoyagi-Ikeda, Kana, Maeno, Toshitaka, Matsui, Hiroki, Ueno, Manabu, Hara, Kenichiro, Aoki, Yasuhiro, Aoki, Fumiaki, Shimizu, Takehisa, Doi, Hiroshi, Kawai-Kowase, Keiko, Iso, Tatsuya, Suga, Tatsuo, Arai, Masashi, Kurabayashi, Masahiko
• Format: Journal Article
• Language: English
• Published: United States American Thoracic Society 01.07.2011
• Subjects: Animals; Antigens, Differentiation - biosynthesis; Cell Line; Cell Movement; Coculture Techniques; Epithelial-Mesenchymal Transition; Fibroblasts - metabolism; Fibroblasts - pathology; Gene Expression Regulation; Male; Myoblasts - metabolism; Myoblasts - pathology; Phosphorylation; Pulmonary Alveoli - metabolism; Pulmonary Alveoli - pathology; Pulmonary Fibrosis - metabolism; Pulmonary Fibrosis - pathology; Rats; Rats, Wistar; Receptors, Notch - metabolism; Response Elements; Signal Transduction; Smad3 Protein - metabolism; Transforming Growth Factor beta1 - metabolism
• ISSN: 1044-1549; 1535-4989
• DOI: 10.1165/rcmb.2010-0140oc

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.