title: Epithelial mesenchymal transition-like phenomenon in trabecular meshwork cells.
abstract: The trabecular meshwork (TM) is a major component of the conventional outflow pathway and the excess extracellular matrix (ECM), and fibrosis in the TM causes increased outflow resistance. In this study, we first investigated the effects of several ECM components in the induction of an epithelial mesenchymal transition (EMT)-like phenomenon in TM cells. TM cells were isolated from cynomolgus monkeys (Macaca fascicularis). The cells were cultured in ECM-coated dishes and then subjected to both western blot analysis and immunocytochemistry to measure the levels of EMT-associated markers. Cell motility was assessed using wound healing and chemotaxis assays. We found that type I collagen, fibronectin and laminin induced the dissociation of cell-cell contact and elongation of actin stress fibers in the cultured monkey TM cells. In addition, following the same stimulation of the ECM, the expression of mesenchymal markers, such as fibronectin and α-smooth muscle actin, and the phosphorylation of Smad2 increased in the TM cells. Our results showed the significant acceleration of TM cellular motility following stimulation with type I collagen, fibronectin and laminin. These phenomena were inhibited by the c-Jun N-terminal kinase (JNK) inhibitor SP600125. In addition, siRNA against paxillin was transfected to evaluate the association between paxillin and the EMT-like phenomenon. The knockdown of paxillin expression by transfection with siRNA blocked the EMT-like alteration of the cellular characteristics and chemotaxis toward transforming growth factor-β2 in the cultured TM cells. Our results showed that the ECM-JNK-paxillin pathway induced an EMT-like phenomenon in TM cells, resulting in the abundant expression of fibronectin and activation of motility in TM cells. This EMT-like phenomenon could result in aberrant conditions in the aqueous outflow pathway in glaucomatous eyes.
authors.affiliation: Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan. Electronic address: eritakahashi@fc.kuh.kumamoto-u.ac.jp.
authors.forename: Eri
authors.initials: E
authors.lastname: Takahashi
authors.affiliation: Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Inoue
authors.affiliation: Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Fujimoto
authors.affiliation: Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Sachi
authors.initials: S
authors.lastname: Kojima
authors.affiliation: Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
authors.forename: Hidenobu
authors.initials: H
authors.lastname: Tanihara
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002465:Cell Movement; D002478:Cells, Cultured; D004195:Disease Models, Animal; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005901:Glaucoma / Q000378:metabolism / Q000473:pathology*; D008252:Macaca fascicularis; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D014129:Trabecular Meshwork / Q000378:metabolism / Q000473:pathology*
chemical_list: D016326:Extracellular Matrix Proteins
keywords: ECM; EMT; JNK; TGF-β; TM; c-Jun N-terminal kinase; epithelial mesenchymal transition; extracellular matrix; integrin ligands; migration; paxillin; trabecular meshwork; trabecular meshwork cell; transforming growth factor-β; α-SMA; α-smooth muscle actin