Abstract
Members of the transforming growth factor beta (TGF-β) superfamily are multifunctional cytokines that regulate several cellular processes, including cell cycle arrest, differentiation, morphogenesis, and apoptosis. TGF-β promotes extracellular matrix production and morphological change. Morphogenetic responses to TGF-β include cell migration and epithelial–mesenchymal transition (EMT), which are critical during embryogenesis, development of fibrotic diseases, and the spreading of advanced carcinomas. The purpose of this study was to clarify how TGF-β regulates the fate of retinal pigment epithelial (RPE) cells. TGF-β1 promoted cell cycle progression and phosphorylation of retinoblastoma protein (Rb) in ARPE-19 cells. TGF-β1 induced survivin expression, which in turn stabilized tubulin and Aurora B. RT-PCR and western blot analysis revealed that survivin expression increased in ARPE-19 cells following TGF-β1 treatment. When survivin was depleted, TGF-β1 induced cell cycle arrest and apoptosis and also reduced Rb phosphorylation. In conclusion, the present study shows that induction of EMT in human RPE cells upregulates survivin, leading to survivin-dependent inhibition of cell cycle arrest and apoptosis. Whether cells undergo EMT or apoptosis in response to TGF-β1 is dependent on their cell cycle state, and TGF-β1 regulates the cell cycle via survivin.
Highlights
Retinal pigment epithelial cells survive during TGF-b1induced epithelial–mesenchymal transition (EMT)
TGF-b1 treatment for 48 h led to dramatic morphological changes and stimulated N-cadherin and fibronectin protein content in the spontaneously immortalized human retinal pigment epithelial (RPE) cell line, ARPE-19 (Figures 1a and b)
To determine whether TGF-b1 induced cell death in human RPE cells, we examined the viability of ARPE-19 cells cultured for 48 h in DMEM containing TGF-b1 in a CCK-8 assay
Summary
Morphogenetic responses to TGF-b isoforms include cell migration and EMT.[2,3,4] EMT is characterized by the disassembly of cell–cell contacts, remodeling of the actin cytoskeleton, and separation of cells, which generates fibroblast-like cells that express mesenchymal markers and migratory properties.[5,6,7,8] TGF-b1 was first described as an inducer of EMT in normal mammary epithelial cells,[9] and has since been shown to mediate EMT in various epithelial cells in vitro, including renal proximal tubular, retinal, lens, and alveolar epithelial cells.[10,11,12,13]. The receptors that mediate TGF-b signaling are well studied. Signaling downstream of TGF-b receptor binding is mediated by Smads, and their interactions have been intensively studied and characterized over the past several years. Crosstalk between different components of the TGF-b signaling pathway may explain the diverse range of effects mediated by TGF-b. Several pathways, involving many downstream proteins, mediate the effects of TGF-b1. Many critical steps in intracellular TGF-b signaling are mediated by Smad proteins. As the Smad pathway principally regulates gene expression, it was originally thought that non-Smad effectors mediate the rapid or direct effects of TGF-b on the actin cytoskeleton.
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