PMID- 25576668 OWN - NLM STAT- MEDLINE DCOM- 20150504 LR - 20150306 IS - 1096-0007 (Electronic) IS - 0014-4835 (Linking) VI - 132 DP - 2015 Mar TI - Negative regulation of TGFbeta-induced lens epithelial to mesenchymal transition (EMT) by RTK antagonists. PG - 9-16 LID - S0014-4835(15)00002-0 [pii] LID - 10.1016/j.exer.2015.01.001 [doi] AB - 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 TGFbeta, 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 TGFbeta-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 TGFbeta-induced EMT over 3 days of culture, as well as alpha-smooth muscle actin (alpha-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 TGFbeta for up to 3 days. The percentages of transfected cells that underwent TGFbeta-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 TGFbeta treatment, with approximately 60% 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 TGFbeta-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 TGFbeta-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. CI - Copyright (c) 2015 Elsevier Ltd. All rights reserved. FAU - Zhao, Guannan AU - Zhao G AD - Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia. FAU - Wojciechowski, Magdalena C AU - Wojciechowski MC AD - Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia. FAU - Jee, Seonah AU - Jee S AD - Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia. FAU - Boros, Jessica AU - Boros J AD - Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia. FAU - McAvoy, John W AU - McAvoy JW AD - Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia; Save Sight Institute, University of Sydney, NSW, Australia. FAU - Lovicu, Frank J AU - Lovicu FJ AD - Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia; Save Sight Institute, University of Sydney, NSW, Australia. Electronic address: frank.lovicu@sydney.edu.au. LA - eng GR - R01 EY03177/EY/NEI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20150107 PL - England TA - Exp Eye Res JT - Experimental eye research JID - 0370707 RN - 0 (Membrane Proteins) RN - 0 (Transforming Growth Factor beta) RN - EC 2.7.10.1 (Receptor Protein-Tyrosine Kinases) SB - IM MH - Animals MH - Blotting, Western MH - Cataract/metabolism MH - Disease Models, Animal MH - Epithelial-Mesenchymal Transition/*drug effects/physiology MH - Lens, Crystalline/*drug effects/physiology MH - Membrane Proteins/metabolism/*physiology MH - Rats MH - Rats, Wistar MH - Receptor Protein-Tyrosine Kinases/*antagonists & inhibitors MH - Reverse Transcriptase Polymerase Chain Reaction MH - Signal Transduction/drug effects MH - Transforming Growth Factor beta/metabolism/*pharmacology OTO - NOTNLM OT - EMT OT - Lens pathology OT - RTK antagonists OT - Sef OT - Spred OT - Spry OT - TGFbeta EDAT- 2015/01/13 06:00 MHDA- 2015/05/06 06:00 CRDT- 2015/01/11 06:00 PHST- 2014/06/02 00:00 [received] PHST- 2014/12/19 00:00 [revised] PHST- 2015/01/06 00:00 [accepted] PHST- 2015/01/11 06:00 [entrez] PHST- 2015/01/13 06:00 [pubmed] PHST- 2015/05/06 06:00 [medline] AID - S0014-4835(15)00002-0 [pii] AID - 10.1016/j.exer.2015.01.001 [doi] PST - ppublish SO - Exp Eye Res. 2015 Mar;132:9-16. doi: 10.1016/j.exer.2015.01.001. Epub 2015 Jan 7.