Negative regulation of TGFß-induced lens epithelial to mesenchymal transition (EMT) by RTK antagonists.
Exp Eye Res
; 132: 9-16, 2015 Mar.
Article
em En
| MEDLINE
| ID: mdl-25576668
ABSTRACT
An eclectic range of ocular growth factors with differing actions are present within the aqueous and vitreous humors that bathe the lens. Growth factors that exert their actions via receptor tyrosine kinases (RTKs), such as FGF, play a normal regulatory role in lens; whereas other factors, such as TGFß, can lead to an epithelial to mesenchymal transition (EMT) that underlies several forms of cataract. The respective downstream intracellular signaling pathways of these factors are in turn tightly regulated. One level of negative regulation is thought to be through RTK-antagonists, namely, Sprouty (Spry), Sef and Spred that are all expressed in the lens. In this study, we tested these different negative regulators and compared their ability to block TGFß-induced EMT in rat lens epithelial cells. Spred expression within the rodent eye was confirmed using RT-PCR, western blotting and immunofluorescence. Rat lens epithelial explants were used to examine the morphological changes associated with TGFß-induced EMT over 3 days of culture, as well as α-smooth muscle actin (α-sma) immunolabeling. Cells in lens epithelial explants were transfected with either a reporter (EGFP) vector (pLXSG), or with plasmids also coding for different RTK-antagonists (i.e. pLSXG-Spry1, pLSXG-Spry2, pLXSG-Sef, pLSXG-Spred1, pLSXG-Spred2, pLSXG-Spred3), before treating with TGFß for up to 3 days. The percentages of transfected cells that underwent TGFß-induced morphological changes consistent with an EMT were determined using cell counts and validated with a paired two-tailed t-test. Explants transfected with pLXSG demonstrated a distinct transition in cell morphology after TGFß treatment, with â¼60% of the cells undergoing fibrotic-like cell elongation. This percentage was significantly reduced in cells overexpressing the different antagonists, indicative of a block in lens EMT. Of the antagonists tested under these in vitro conditions, Spred1 was the most potent demonstrating the greatest block in TGFß-induced fibrotic cell elongation/EMT. Through the overexpression of RTK-antagonists in lens epithelial cells we have established a novel role for Spry, Spred and Sef as negative regulators of TGFß-induced EMT. Further investigations may help us develop a better understanding of the molecular mechanisms involved in maintaining the integrity of the normal lens epithelium, with these antagonists serving as putative therapeutic agents for prevention of EMT, and hence cataractogenesis.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Fator de Crescimento Transformador beta
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Receptores Proteína Tirosina Quinases
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Transição Epitelial-Mesenquimal
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Cristalino
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Proteínas de Membrana
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Exp Eye Res
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Austrália