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Ayako Fukushima, Eri Takahashi, Tomokazu Fujimoto, Sachi Kojima, Toshihiro Inoue, Hidenobu Tanihara; Type I collagen-induced Epithelial Mesenchymal Transition-like Phenomenon in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3557.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the effects of type I collagen (Col I) on monkey trabecular meshwork (TM) cells.
TM cells were cultured on Col I-coated or uncoated dish in serum-free medium for 48 hours. Cell lysates were subjected to western blot assays with antibodies against epithelial or mesenchymal markers. Cell-cell adhesion was stained for β-catenin and images were gained by confocal fluorescence microscopy. The migratory ability was evaluated by the wound healing assay. Confluent monolayer was scratched by sterile tip and photographed at each time, and evaluated by the measuring the migration from the edge. The blots were quantified using Image J.
TM cells showed epithelial-like island appearance on uncoated dish, and the same cells on Col I-coated dish demonstrated scattering phenotype. Western blot analysis showed the increased expression of mesenchymal markers (fibronectin (p=0.015), α-SMA (p=0.043), and phospho Smad2 (p=0.027)) induced by Col I. In contrast, similar experiments revealed decreased expression of an epithelial marker, occludin, under the stimulatin of Col I. Confocal images of staining with β-catenin revealed linear staining at the cell-cell borders in TM cells on uncoated dish, and Col I-coating disrupted it. In addition, Col I enhanced cell motility in TM cells compared to controls (uncoated dish) (p=0.0053). Further, JNK inhibitor, SP600125, inhibited Col I-induced changes of mesenchymal phenotype, up-regulation of fibronectin (p=0.036) and high motility (p=0.0021).
Our findings indicated that the occurrence of EMT-like phenomenon caused by extracellular matrix in TM cells, suggesting the contribution of this phenomenon to the aberrant status in aqueous humor flow of glaucomatous eyes.
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