title: Notch induces myofibroblast differentiation of alveolar epithelial cells via transforming growth factor-{beta}-Smad3 pathway.
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.
authors.affiliation: Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8511, Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Aoyagi-Ikeda
authors.forename: Toshitaka
authors.initials: T
authors.lastname: Maeno
authors.forename: Hiroki
authors.initials: H
authors.lastname: Matsui
authors.forename: Manabu
authors.initials: M
authors.lastname: Ueno
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Hara
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Aoki
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Aoki
authors.forename: Takehisa
authors.initials: T
authors.lastname: Shimizu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Doi
authors.forename: Keiko
authors.initials: K
authors.lastname: Kawai-Kowase
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Iso
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Suga
authors.forename: Masashi
authors.initials: M
authors.lastname: Arai
authors.forename: Masahiko
authors.initials: M
authors.lastname: Kurabayashi
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis; D002460:Cell Line; D002465:Cell Movement; D018920:Coculture Techniques; D058750:Epithelial-Mesenchymal Transition; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D008297:Male; D032446:Myoblasts / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D011650:Pulmonary Alveoli / Q000378:metabolism* / Q000473:pathology; D011658:Pulmonary Fibrosis / Q000378:metabolism / Q000473:pathology; D051381:Rats; D017208:Rats, Wistar; D051880:Receptors, Notch / Q000378:metabolism*; D020218:Response Elements; D015398:Signal Transduction; D051900:Smad3 Protein / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
chemical_list: D000943:Antigens, Differentiation; D051880:Receptors, Notch; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D053773:Transforming Growth Factor beta1