Negative regulation of TGFb-induced lens epithelial to mesenchymal transition (EMT) by RTK antagonists Guannan Zhao a , Magdalena C. Wojciechowski a , Seonah Jee a , Jessica Boros a , John W. McAvoy a, b , Frank J. Lovicu a, b, * a Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia b Save Sight Institute, University of Sydney, NSW, Australia article info Article history: Received 2 June 2014 Received in revised form 19 December 2014 Accepted in revised form 6 January 2015 Available online 7 January 2015 Keywords: TGFb Lens pathology EMT RTK antagonists Spry Sef Spred 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 TGFb, 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 TGFb-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 TGFb-induced EMT over 3 days of culture, as well as a-smooth muscle actin (a-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 TGFb for up to 3 days. The percentages of transfected cells that underwent TGFb- 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 TGFb treatment, with ~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 TGFb-induced fibrotic cell elongation/EMT. Through the over- expression of RTK-antagonists in lens epithelial cells we have established a novel role for Spry, Spred and Sef as negative regulators of TGFb-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. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction The establishment and maintenance of the distinctive archi- tecture of the mammalian lens, and hence its transparency, is regulated by the ocular environment, specifically the ocular fluids that bathe the lens, the aqueous and vitreous humor (Lovicu and McAvoy, 2005). Many different growth factors found in this ocular media, such as FGF, regulate normal lens cellular processes, sustaining lens growth throughout life, while other factors, such as TGFb, especially if deregulated, can induce aberrant lens cell behavior leading to a lens epithelial-to-mesenchymal transition (EMT) that underlies both anterior subcapsular cataract (ASC) and the most common complication after cataract surgery, posterior capsular opacification (PCO; Liu et al., 1994; Srinivasan et al., 1998; Lovicu et al., 2002; de Iongh et al., 2005). More recent studies from our laboratory have shown that murine lens epithelial cells * Corresponding author. Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia. E-mail address: frank.lovicu@sydney.edu.au (F.J. Lovicu). Contents lists available at ScienceDirect Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer http://dx.doi.org/10.1016/j.exer.2015.01.001 0014-4835/© 2015 Elsevier Ltd. All rights reserved. Experimental Eye Research 132 (2015) 9e16