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Negative regulation of TGFβ-induced lens epithelial to mesenchymal transition (EMT) by RTK antagonists
Experimental Eye Research 132 (2015) 9e16
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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 b
Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia Save Sight Institute, University of Sydney, NSW, Australia
a r t i c l e i n f o
a b s t r a c t
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
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 RTKantagonists (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 TGFbinduced 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 overexpression 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.
Keywords: TGFb Lens pathology EMT RTK antagonists Spry Sef Spred
1. Introduction The establishment and maintenance of the distinctive architecture 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
* Corresponding author. Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia. E-mail address: [email protected] (F.J. Lovicu). http://dx.doi.org/10.1016/j.exer.2015.01.001 0014-4835/© 2015 Elsevier Ltd. All rights reserved.
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
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deficient for Sprouty (Spry) are more sensitive to this TGFb-induced insult, leading to aberrant cell behavior, EMT and subsequent cataract (Shin et al., 2012). Sef, Spry and Spred (Sprouty-related protein) belong to families of negative-regulatory molecules reported to act on receptor tyrosine kinases (RTKs), including the FGF-Ras-ERK/MAPK pathway (Hacohen et al., 1998; Casci et al., 1999; Kramer et al., 1999; Chambers and Mason, 2000; Wakioka et al., 2001; Li et al., 2003; Ozaki et al., 2005). Spry and Sef are normally expressed in the lens and eye throughout embryogenesis and postnatal growth (Boros et al., 2006), consistent with them playing an important role during early lens and eye development (Newitt et al., 2010; Kuracha et al., 2011, 2013), as well as in other tissues and organs (Kramer et al., 1999; Tefft et al., 1999; Chambers and Mason, 2000; Boros et al., 2006; Mason et al., 2006). Although a deficiency of Sef in the lens does not appear to have an impact on lens development (see Newitt et al., 2010), when Spry1 and Spry2 are conditionally deleted from the murine lens, postnatally, the lens epithelia of these mutant mice undergo aberrant cell behavior leading to cataract (Shin et al., 2012). This deficiency of Spry in the lens was shown to impair lens epithelial intracellular signaling, including increased phosphorylation of ERK1/2, as well as nuclear localization of Smad2/3, snail and slug (downstream targets of TGFbsignaling) that lead to deregulation of E-cadherin and upregulation of alpha-smooth muscle actin (a-sma), all leading to transdifferentiation of cells into myofibroblastic cells with aberrant deposition of extracellular matrix, defining an EMT (Shin et al., 2012). More convincingly, overexpression of Spry in lens epithelia, both in vitro and in situ, was shown to effectively block the influence of TGFb, as evident by the repression of nuclear localization of phosphorylated Smads, snail and slug, as well as a block in a-sma accumulation, and maintenance of E-cadherin (Shin et al., 2012). In situ, this translated to Spry effectively preventing/ blocking TGFb-induced EMT and cataract. Based on these studies we proposed that RTK antagonists, such as Spry, may play an important role in the maintenance of the normal monolayer of lens epithelia, protecting it to some degree from the detrimental effects of deregulated TGFb signaling. Moreover, for TGFb to elicit its effects on the lens, we would expect that Spry itself would need to be downregulated, and indeed this is the case, supported by gene array studies demonstrating that Spry1 and Spry2 levels are both reduced in lens cells (FLH124) exposed to TGFb (Dawes et al., 2007). To better understand the role of the different RTK antagonists expressed in the lens, we screened other putative negative regulators of TGFb-mediated EMT, using the lens epithelial explant system. For this we examined and compared different RTK inhibitors including Sprouty1, Sprouty2 and Sef, as well as members of the Spred family, Spred1, Spred2 and Spred3; that we show here to also be expressed normally in the eye and lens cells. Using this approach, we demonstrate that all the antagonists tested can reduce the ability of TGFb to induce an EMT in lens epithelial cells. This data supports a key role for negative regulators of intracellular signaling, to protect lens cells not only from the adverse effects of compounds such as TGFb, but also to maintain lens cell integrity and hence transparency. 2. Material and methods All animal handling and operating procedures carried out in this study adhered to the ARVO statement for the use of animals in ophthalmic research, conforming to the provisions of the code of practice provided by the National Health and Medical Research Council (NHMRC, Australia), and were approved by the Animal Ethics Committee of the University of Sydney, NSW, Australia. Ocular tissues were derived from postnatal day 3 (P3), P15 and P21
albino Wistar rats. 2.1. RT-PCR Unless otherwise stated, steps utilizing commercial reagents and kits were carried out according to manufacturer instructions. TriReagent (Molecular Research Centre, OH, USA) was used to extract total RNA from the following tissues harvested from P15 rats: whole eye, whole lens, lens capsule (with adherent epithelial cells), and lens fibers. To remove DNA that may have co-purified with the RNA, each sample was incubated with DNase I (Amersham Biosciences, NJ, USA) for 30 min at 37 C. Treatment with DNase Inactivation Reagent (Life Technologies, CA, USA) was carried out to remove the enzyme and divalent cations that would otherwise be detrimental to downstream DNA synthesis. First strand cDNA was reverse transcribed from 2 mg of the prepared total RNA using Promega's Reverse Transcription System (WI, USA). Control reactions omitting AMV reverse transcriptase, were set up and later tested, confirming that genomic DNA was absent from the purified RNA template (data not shown). To investigate Spred expression, gene specific primers for coding regions of Spred1 (50 agacacagcctccctcgtta-30 , 50 -ttatgtttcccaccacagca-30 ), Spred2 (50 cgggtgagctgtatgtggt-30 , 50 -atacaaggggccaggaaaga-30 ) and Spred3 (50 -cggatgagttccagaagagc-30 , 50 -gatcgccggtagtcctcata-30 ) were incorporated into PCR reactions containing 5 ml cDNA, 1.7 mM MgCl2, 1 mM dNTPs, 1x NH4 reaction buffer and 0.8 U BioTaq Red™ (Bioline, London, UK). Reactions were denatured at 94 C for 3 min, amplified through 32 cycles (94 C for 30 s, 58 C for 30 s, 72 C for 1 min) and allowed a final extension at 72 C for 2 min. The end PCR products were analyzed by agarose gel electrophoresis. 2.2. SDS-PAGE and western blotting Lens epithelial cells and fiber cell samples were collected from P15 rats and homogenized in cold lysis buffer containing 2.5 mM EDTA, 25 mM TriseHCl (pH 7.5), 0.375 M NaCl, 1% IGEPAL, 1.5 mM sodium orthovanadate and a protease inhibitor cocktail (complete, Mini; Roche, Basel, Switzerland). To facilitate lysis, the homogenized samples were rotated for 2 h at 4 C. Protein quantification using the Micro-BCA protein assay kit (Pierce, IL, USA) enabled size separation of 20 mg of total protein through a 10% SDS-PAGE matrix. Prior to gel loading, protein lysates were mixed in a 1:1 ratio with Laemmli sample buffer (BioRad, CA, USA) containing 5% (v/v) 2mercaptoethanol as a denaturant. Once separated, proteins were transferred from the gel matrix to a PVDF membrane (Millipore). The membrane was incubated (1 h at RT) with a blocking solution of 5% (w/v) skim milk powder in TBS-T (0.1% (v/v) Tween 20 in trisbuffered saline (TBS) and then probed overnight with polyclonal antibodies generated from GST fusion proteins of human Spred1, 2 or 3 (diluted 1:1000 in blocking solution). Antibody specificity was verified by Western blot analysis of transfected HEK-293T cell lysates (see King et al., 2005). Unbound antibody was removed with multiple TBS-T washes, before a 2 h incubation with goat antirabbit HRP-conjugated IgG (diluted 1:5000 in TBS-T; Cell Signaling Technologies, MA, USA). Again, unbound antibody was removed with TBS-T washes, this time followed by application of Immobilin Western Chemiluminescent HRP substrate (Millipore). Hyperfilm™ (Amersham Biosciences) was used to capture the chemiluminescence, which was visualized using standard autoradiography techniques. 2.3. Immunofluorescent labeling of explants and whole-mounts Lens epithelial explants were prepared as described previously (Lovicu and McAvoy, 2008), and were cultured in Medium 199 with
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Earle's salts (M199; Trace Scientific, NSW, Australia), supplemented with 0.1% bovine serum albumin (BSA; Sigma, NSW, Australia), 0.1 mg/ml L-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml Amphostat (all from Trace Scientific, Australia) at 37 C in 5% CO2. Explants were cultured with 100 pg/ml TGFb-2 (R&D Systems, MN, USA) for 3 days. Prior to immunoflourescent labeling, explants were viewed with phase-contrast microscopy (CK2, Olympus, Japan) and photographed (DFC-280, Leica, Germany). Lens epithelial wholemounts or explants were fixed with 100% methanol for 45 s and rinsed in succession with PBS. Following rinses (3 5 min) with 0.1% (w/v) BSA in PBS (PBS/BSA), explants were incubated with a 10% normal goat serum (NGS) blocking agent (1 h at RT). Excess blocking buffer was removed and anti-Spred antibodies (described above) or mouse-anti-a-sma (1:100 dilution in 1.5% NGS; Sigma, NSW, Australia) was applied to the explants for an overnight incubation at 4 C. Excess primary antibody was removed with rinses in PBS/BSA (3 5 min), prior to the application of secondary antibody. Antimouse- or anti-rabbit-Alexa 488 or Alexa-594 (all antibodies diluted 1:1000 in PBS/BSA; Invitrogen, USA) were applied to explants and left for 2 h at room temperature. Cell nuclei were visualized with Hoechst dye (6 mg/ml; Calbiochem, USA) for 5 min. Following 3 5 min washes in PBS/BSA, the explants were mounted with 10% PBS (v/v) in glycerol and immunofluorescent labeling was visualized and imaged using confocal microscopy (Zeiss LSM 700). 2.4. DNA transfection of lens epithelial explants
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buffered formalin (NBF) for 20 min, and rinsed with 70% ethanol (3 5 min). For lectin staining of lens cell membranes, explants were rehydrated in 0.1% (w/v) BSA in PBS (PBS/BSA; 3 5 min washes) at room temperature. The explants were then permeabilized in 0.05% Tween-20 in PBS/BSA (3 5 min), and rinsed in PBS/BSA (2 5 min). After wiping off excess fluid, lectin-TRITC (1:100 dilution in PBS/BSA; Sigma, NSW, Australia) was applied directly onto the explant for 1 h at room temperature. Explants were then rinsed with PBS/BSA (3 5 min) prior to nuclear labeling with Hoechst dye (Calbiochem) for 5 min. Explants were mounted in 10% PBS (v/v) in glycerol and visualized using confocal microscopy (Zeiss LSM 700). 3. Results 3.1. Spred expression in the eye Using P15 Wistar rat ocular tissues, RT-PCR demonstrated that all Spreds; Spred1, Spred2 and Spred3, were expressed in the eye (Fig. 1A). This ocular expression, at least in part, was due to the expression of Spred in the intact lens, with stronger expression demonstrated in the lens epithelium, compared to the lens fiber cells. The lens expression translated to the presence of the respective Spred proteins in lysates of corresponding lens epithelia (Fig. 1B). In P3 lens/eye tissue sections we see strong immunofluorescent labeling for Spreds, for example Spred-1 in epithelia of the cornea, ciliary body and lens, as well as in the lens fibers (Fig. 2). Strong labeling was seen at the epithelial to fiber cell interface
The construction of DNA plasmids of interest (pLXSG, pLXSGSprouty1, pLXSG-Sprouty2, pLXSG-Sef, pLXSG-Spred1, pLXSGSpred2 and pLXSG-Spred3) were confirmed by sequencing and extracted and purified according to manufacturer's instructions (Astral scientific, Australia). Using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA), 2 mg of DNA plasmid encoding for the antagonist of interest was obtained. Lipofectamine 2000 (Invitrogen, USA) was diluted in unsupplemented M199 media (1:10 dilution), and mixed with 2 mg of DNA and kept at room temperature for 20 min to form the DNA-Lipofectamine complexes. The DNA-Lipofectamine complexes were added to culture dishes containing epithelial explants (described above) and incubated at 37 C, 5% CO2 for 48 h. To confirm the successful transfection of the explants, the expression of enhanced-GFP was visualized using an inverted fluorescent microscope (Leica DMLB, Germany). Culture media was replaced with freshly supplemented M199 medium, and explants were then treated with 100 pg/ml TGFb-2 for up to 3 days. 2.5. Quantification of TGFb-induced EMT The successfully transfected lens epithelial cells expressing EGFP were photographed after 3 days of culture. Cell counts were carried out in a blind test to eliminate bias. An epithelial cell was defined as being cuboidal in shape, similar to epithelial cells prior to the addition of TGFb. A cell that elongated and was fibrotic in appearance, extending cellular processes beyond the normal boundaries of an epithelial cell was considered to have undergone an EMT. The percentage of labeled cells undergoing an EMT was determined and statistical significance was inferred using a paired two tailed t-test. 2.6. Lectin staining of transfected lens epithelial explants Transfected lens epithelial explants were fixed with 10% neutral
Fig. 1. (A) RT-PCR was used to detect Spred expression in postnatal day 15 rat eye (lane 1), lens (lane 2), lens epithelium (lane 3) and lens fibers (lane 4). A negative control, no DNA sample (lane 5) and positive control, Spred plasmid (lane 6) and size markers (lane 7) are also shown. Spred1, Spred2 and Spred3 are expressed in whole eye, with expression of Spred1-3 in lens epithelia and Spred1 and Spred2 in lens fibers. (B) Western blotting also demonstrated Spred1-3 protein in lens epithelial cell lysates. atubulin was used as a loading control.
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Fig. 2. Representative immunofluorescent labeling for Spred (Spred-1) in ocular sections of neonatal (P3) rats (C,D), counterstained with Hoechst dye for nuclei (A,B). Spred-1 is strongly expressed in both lens epithelial cells (le) and fiber cells (lf), with strong labeling at their interface (C,D, arrows). Strong labeling was also observed in corneal epithelia (ce) as well as developing ciliary body and iris. Whole mounts of lens epithelia also demonstrated membrane labeling for Spred-1 in fiber cells (E, green) and some cytoplasmic labeling in epithelial cells (F). Note that in whole mounts of lens epithelium, patches of adherent apical tips of fibers are frequently retained and can be visualised in optical sections. Scale Bar, 20 mm.
(Figure AeD, arrows), which was also highlighted in lens epithelial whole-mounts, demonstrating membrane labeling at the apical tips of fiber cells (Fig. 2E), as well as cytoplasmic labeling in many of the epithelial cells (Fig. 2F). 3.2. TGFb-induced epithelial-to-mesenchymal transition (EMT) Lens epithelial explants treated with or without TGFb were collected after 3 days of culture, and any morphological changes induced in the epithelial cells were initially observed using phasecontrast microscopy. Explants were immunolabeled for a-sma. The presence of TGFb induced a distinctive transition in cell morphology over the 3 days of culture, with the lens epithelial cells undergoing an EMT characterized by cell elongation into spindlelike cells (Fig. 3B, arrows). There was also evidence of cell loss due to apoptosis (Fig. 3B, asterisk; see Maruno et al., 2002) with distinct regions devoid of cells, exposing the lens capsule that displayed capsular wrinkling (Fig. 3B, arrowheads). Explants cultured with TGFb demonstrated strong labeling for a-sma in distinct stress fibers, consistent with a mesenchymal phenotype (Fig. 3D). In contrast, control explants showed no notable cellular changes over the culture period with the monolayer of epithelial cells retaining their cuboidal morphology (Fig. 3A). Consistent with this, the control cells expressed basal levels of amorphous a-sma labeling (Fig. 3C). 3.3. RTK-antagonists block TGFb-induced EMT To examine the efficiency of growth factor antagonists in preventing TGFb-induced EMT, cells in lens epithelial explants were transfected with a panel of different antagonists normally expressed in the lens (Spry1, Spry2, Sef, Spred1, Spred2 and Spred3). Successful transfection was evident by the expression of
reporter protein (EGFP), indicative of plasmid uptake by the cells (Fig. 4). Live transfected cells were viewed and imaged, and cell counts were used to quantify the percentage of cells undergoing fibrotic cell changes induced by TGFb. Using lectin membrane labeling we could observe the transfected cells in the context of the surrounding non-transfected cells within the explant. Fig. 4 shows representative results for the Spry2 and Spred3 transfection studies as examples of the effects of the different treatments. When explants were transfected with pLXSG alone (reporter plasmid) and cultured without TGFb for 3 days (Fig. 4A, E), the EGFP-expressing (labeled) cells maintained their irregular cuboidal shape, indicating that EGFP expression has no direct influence on lens epithelial cell integrity. However, when these same cells were treated with TGFb (Fig. 3B, F), the pLXSG transfected cells elongated by 3 days, typical of a TGFb-induced fibrotic/EMT response. This was consistent with the surrounding non-EGFP expressing cells (Fig. 4F), demonstrating that EGFP expression alone is not sufficient to inhibit TGFb-induced EMT. On average, 61% of the cells in explants that were transfected with pLXSG alone and treated with TGFb underwent fibrotic-like cellular changes (Fig. 5). With the addition of TGFb, the morphology of epithelial cells that were not transfected (and thus not expressing EGFP) transitioned normally into a mesenchymal phenotype and exhibited cell loss consistent with a TGFb-induced EMT response (Fig. 4FeH). Given the relatively low transfection efficiency of our primary explants, we could not effectively assay or quantify for changes in any molecular markers under these conditions; however, when we doublelabeled for EGFP-expressing transfected cells (for example, overexpressing Spry2) with a-sma, we noticed a reduced staining for asma in these cells that failed to undergo an EMT in response to TGFb. Compared to non-transfected neighboring cells displaying a more prominent mesenchymal phenotype, a-sma was weaker and mostly diffuse (not localized to stress fibers) in transfected cells
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Fig. 3. TGF-b induced EMT after 3 days. (A) Phase contrast microscopy of explants cultured without TGFb show the tightly packed monolayer of cuboidal cells. (B) When explants were cultured with 100 pg/ml of TGFb for 3 days, cell elongation (arrows), exposed regions of lens capsule (asterisk) and capsular wrinkling (arrowheads) are evident. (C) Immunoflourescent labeling showed that explants cultured without TGFb exhibited low levels of amorphous a-sma labeling in the cytoplasm (green). Hoechst nuclear counterstaining (red) demonstrates rounded nuclei consistent with an epithelial phenotype. (D) Explants cultured with TGFb exhibit distinct a-sma immunoreactivity in stress fibers, with nuclear staining revealing more elongate and irregular shaped nuclei, consistent with a mesenchymal phenotype.
that failed to undergo an EMT in response to TGFb (see Fig. 6). The overexpression of RTK-antagonists was not sufficient to initiate cellular changes associated with EMT without the presence of TGFb. Explants retained their tightly packed, cuboidal monolayer under these conditions (data not shown). When cells were transfected with Spry1 and Spry2 there was a significant decrease in the percentage of cells elongating and undergoing a TGFb-induced EMT, compared to control explants (p < 0.001). A 64% reduction in labeled cells demonstrating fibrotic changes resulted when epithelial cells were transfected with pLXSG-Spry1 and exposed to TGFb (Fig. 5). Similarly, when transfected with pLXSG-Spry2, 71% of the labeled lens epithelial cells did not undergo TGFb-induced EMT. The overexpression of Sef (pLXSG-Sef) led also to a significant inhibition of TGFb-induced EMT, with 76% (p < 0.001) of transfected/ labeled cells remaining cuboidal when cultured with TGFb. The Spred family of RTK-antagonists, of which we demonstrate here to be also present in the lens, appear to be the most potent inhibitors of TGFb-induced EMT. Transfection of lens epithelial cells with pLXSG-Spred1 significantly decreased TGFb-induced fibrotic changes by 92% compared to control explants (p < 0.001). The overexpression of Spred2 (pLXSG-Spred2) resulted in an 82% reduction, whereas with Spred3, a 91% reduction; a significant
inhibition of TGFb-induced EMT in all cases (p < 0.001). In summary, the overexpression of RTK-antagonists in the presence of TGFb leads to a significant reduction of the TGFbassociated cellular changes; however, it does not lead to a complete block in the TGFb-induced EMT. Most cells in explants that were transfected with RTK-antagonists and treated with TGFb retained the typical cuboidal epithelial shape (Fig. 4G, H), while others (albeit a smaller percentage) were still capable of undergoing an EMT. 4. Discussion Transforming growth factor b (TGFb) induces an epithelial-tomesenchymal transition (EMT) that results in lens opacities characteristic of those found in some forms of human cataract (see De Iongh et al., 2005). Accordingly, the actions of TGFb are tightly regulated in normal lens cell biology. One mode of regulation is proposed to be via intracellular antagonists reported to regulate RTK-induced intracellular signaling pathways. Antagonists of the MAPK/ERK signaling pathway such as Spry and Sef have been shown to be strongly expressed in the lens epithelium (Boros et al., 2006; Shin et al., 2012) and we show here that other antagonists
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Fig. 4. DNA transfection with different RTK antagonists inhibits TGFb induced EMT. Transfected cells express EGFP (green). Without TGFb, pLXSG-expressing cells retained a typical irregular cuboidal shape (A), consistent with the surrounding cells visualised by lectin staining (E). When cultured with TGFb for 3 days, pLXSG expressing cells and surrounding cells underwent EMT and elongated (B, F). Cells transfected with pLXSG-Spry (C, G) and pLXSG-Spred (D, H), retained a cuboidal shape, whereas surrounding cells elongated and underwent an EMT as evident by cell loss and areas of bare capsule.
Fig. 5. Comparison of the percentage of TGF-b-induced EMT in lens epithelia cells overexpressing the RTK-antagonists of interest (Spry1, Spry2, Sef, Spred-1, Spred-2 and Spred-3) or only the reporter gene (pLXSG). A significant reduction of TGFb-induced change in morphology is evident in the presence of the antagonists (n ¼ 20 for each RTK-antagonist). *P < 0.001.
including Spred1, Spred2 and Spred3 are also expressed in the lens. The current investigation provides a first screen of these selective MAPK/ERK1/2 antagonists, Spry, Sef and Spred to determine how effective they are at blocking TGFb-induced EMT. All antagonists examined were shown to have the ability to block TGFb-induced
EMT, with Spred1 in particular found to be the most potent antagonist in this system. This may be attributed to the larger Nterminal Enabled/VASP homology 1 (EVH1) domain of Spred1 (McClatchey and Cichowski, 2012), which may confer an increased ability to inhibit, compared to other Spreds, for example (King et al.,
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Fig. 6. TGFb-induced EMT after 3 days. Immunoflourescent labeling showed that cells in lens explants cultured with TGFb exhibited distinct a-sma immunoreactivity, notably in stress fibers (red, arrows; C,D), consistent with a mesenchymal phenotype; however, transfected cells co-labeled for EGFP (B,D; overexpressing Spry2 in this instance) exhibited lower levels of a more amorphous a-sma-labeling in the cytoplasm (arrowhead). Hoechst dye counterstaining (blue; A,D) highlights the cell nuclei. Scale Bar, 50 mm.
2005). TGFb has a potent cataractogenic effect on lens epithelial cells, disrupting the monolayer of cuboidal cells and cellular integrity, resulting in a morphological transition into elongate myofibroblastic/fibroblastic cells. These cells also express a-sma in response to TGFb, a hallmark of EMT. Whilst were able to demonstrate that transfected cells failing to undergo an EMT in response to TGFb had reduced levels of a-sma, we would require a greater cell transfection efficiency to quantify this. We did however note that a-sma in these cells was not distributed in stress fibers, typical of myofibroblastic cells as shown in neighboring non-transfected cells responding to TGFb. We must be mindful that the RTK antagonists overexpressed may be uncoupling the EMT process by only targeting select signaling pathways impacting on cell morphology, and not all signaling (downstream of TGFb) leading to an EMT that may involve a-sma accumulation. Although all the antagonists tested were shown to potentially block TGFb-induced cellular responses in this system, the presence of both cuboidal epithelial cells, that resisted undergoing an EMT, as well as cells that still retained the ability to undergo morphological changes typical of an EMT, highlights the different efficiency of the individual antagonists. This variability in effectiveness of each antagonist may also depend upon the level of antagonist expression within specific cells. This is a variable of our in vitro system that we could not control. Nevertheless, overexpression studies in situ provide further validation of the antagonistic effects of the RTKinhibitors on the TGFb-induced cellular responses. For example, some of our recent studies have shown that overexpression of Spry
in transgenic mice can ameliorate anterior subcapsular cataract (ASC) induced by TGFb overexpression (Shin et al., 2012). Consistent with this, knock-out studies of Spry proteins in the murine lens result in ASC plaque formation and increases in TGFb signaling, including induced nuclear localization of Smad2/3, snail and slug, similar to that found in mice overexpressing TGFb (Shin et al., 2012). Given that Spry proteins are required for lens epithelial integrity and have a profound effect on TGFb-induced cataract in situ, as well as the ability to significantly hamper the TGFb-inducedEMT when overexpressed through the transfection of lens epithelial explants, it is clear that the Spry family of proteins have a crucial role in the regulation of the TGFb-induced EMT. Sef has also been shown to be strongly expressed in the lens epithelium during embryogenesis and postnatally (Boros et al., 2006). The present study is the first to examine the role of Sef protein in the context of TGFb-induced EMT of the lens. It was shown that the overexpression of Sef is more effective than both Spry1 and Spry2 at blocking the TGFb-induced cellular responses within explants. Given that Sef has been proposed to block FGF signaling in lens (Newitt et al., 2010), as well as TGFb-induced EMT in our system, it would be interesting to further examine the role FGF plays in TGFb-induced EMT. FGF has previously been shown to induce ERK1/2 signaling (Lovicu and McAvoy, 2001), a primary target of these antagonists (Hanafusa et al., 2002; Torri et al., 2004; Quintanar-Audelo et al., 2011). In other systems, such as rat mesangial cells (Jiang et al., 2010) and mouse cortical tubule cells (Xie et al., 2004), TGFb has been shown to signal through Smadindependent signaling such as the MAPK/ERK pathway during
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EMT. The putative cross-talk between the MAPK/ERK and canonical TGFb signal transduction pathways identifies a possible mechanism for these growth factor antagonists to inhibit TGFb-induced EMT. As previous studies have shown Spry, Sef and Spred to antagonize the MAPK/ERK signaling pathway (Hanafusa et al., 2002; Torri et al., 2004; Quintanar-Audelo et al., 2011), it is likely that these specific RTK-antagonists are exerting their actions on MAPK/ERK signal transduction to block TGFb-induced lens EMT. Further studies to determine how these RTK-antagonistic molecules slow or block the progression of subcapsular plaque formation, and the role for ERK1/ 2, will provide greater insights into the mechanism of TGFbmediated cataract formation in situ. In summary, it is clear that these antagonists impede the fibrotic cellular changes associated with TGFb-induced EMT in situ (for Spry) and now within lens epithelial explants. Spry, Sef and Spred may therefore serve as putative therapeutic agents for the prevention of EMT, and thus cataractogenesis. Acknowledgments The authors would like to thank A/Prof Christopher Hovens (University of Melbourne, Vic, Australia) for providing the Spred antibodies. The work was funded by the NIH (R01 EY03177), USA, the National Health and Medical Research Council, and the Sydney Foundation for Medical Research, Australia. References Boros, J., Newitt, P., Wang, Q., Mcavoy, J.W., Lovicu, F.J., 2006. Sef and sprouty expression in the developing ocular lens: implications for regulating lens cell proliferation and differentiation. Seminars Cell Dev. Biol. 17, 741e752. Casci, T., Vinos, J., Freeman, M., 1999. Sprouty, an intracellular inhibitor of Ras signaling. Cell 96, 655e665. Chambers, D., Mason, I., 2000. Expression of sprouty2 during early development of the chick embryo is coincidet with known sites of FGF signalling. Mech. Dev. 91, 361e364. Dawes, L.J., Elliot, R.M., Reddan, J.R., Wormstone, Y.M., Wormstone, I.M., 2007. Oligonucleotide microarray analysis of human lens epithelial cells: TGFb regulated gene expression. Mol. Vis. 1181e1197. De Iongh, R.U., Bellusci, S., Lovicu, F.J., Mcavoy, J.W., 2005. Transforming growth factor-b induced epithelial-mesenchymal transition in the lens: a model for cataract formation. Cells Tissues Organs 179, 43e55. Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y., Krasnow, M.A., 1998. Sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92, 253e256. Hanafusa, H., Torri, S., Yasunaga, T., Nishida, E., 2002. Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signaling pathway. Nat. Cell. Biol. 4, 850e858. Jiang, W., Zhang, Y., Wu, H., Zhang, X., Gan, H., Sun, J., Chen, Q., Guo, M., Zhang, Z., 2010. Role of cross-talk between the Smad2 and MAPK pathways in TGF-b1induced collagen IV expression in mesangial cells. Int. J. Mol. Med. 26, 571e576. King, J.A.J., Straffon, A.F.L., D'Abaco, G.M., Poon, C.L.C., Stacey, T.T.I., Smith, C.M., Buchert, M., Corcoran, N.M., Hall, N.E., Callus, B.A., Sarcevic, B., Martin, D., Lock, P., Hovens, C.M., 2005. Distinct requirements for the sprouty domain for
functional activity of spred proteins. Biochem. J. 388, 445e454. Kramer, S., Okabe, M., Hacohen, N., Krasnow, M.A., Hiromi, Y., 1999. Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila. Development 126, 2515e2525. Kuracha, M.R., Burgess, D., Siefker, E., Cooper, J.T., Licht, J.D., Robinson, M.L., Govindarajan, V., 2011. Spry1 and Spry2 are necessary for lens vesicle separation and corneal differentiation. Investigative Ophthalmol. Vis. Sci. 52, 6887e6897. Kuracha, M.R., Siefker, E., Licht, J.D., Govindarajan, V., 2013. Spry1 and Spry2 are necessary for eyelid closure. Dev. Biol. 383, 227e238. Li, J., Xia, F., Li, W.X., 2003. Coactivation of STAT and Ras is required for germ cell proliferation and invasive migration in Drosophila. Dev. Cell. 5, 787e798. Liu, J., Hales, A.M., Chamberlain, C.G., McAvoy, J.W., 1994. Induction of cataract-like changes in rat lens epithelial explants by transforming growth factor-b. Investigative Ophthalmol. Vis. Sci. 35, 388e401. Lovicu, F.J., McAvoy, J.W., 2001. FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling. Development 128, 5075e5084. Lovicu, F.J., McAvoy, J.W., 2005. Growth factor regulation of lens development. Dev. Biol. 280, 1e14. Lovicu, F.J., Schulz, M.W., Hales, A.M., Vincent, L.N., Overbeek, P.A., Chamberlain, C.G., Mcavoy, J.W., 2002. TGFbeta induces morphological and molecular changes similar to human anterior subcapsular cataract. Br. J. Ophthalmol. 86, 220e226. Lovicu, F.J., McAvoy, J.W., 2008. Epithelial explants and their application to study developmental processes in the lens. In: Tsonis, P.A. (Ed.), Animal Models in Eye Research. Elsevier, Academic Press, pp. 134e147. Maruno, K., Lovicu, F.J., Chamberlain, C.G., McAvoy, J.W., 2002. Apoptosis is a feature of TGFß-induced cataract. Clin. Exp. Optometry 85, 76e82. Mason, J.M., Morrison, D.J., Basson, M.A., Licht, J.D., 2006. Sprouty proteins: multifaceted negative-feedback regulators of receptor tyrosine kinase signaling. Trends Cell Biol. 16, 45e54. Newitt, P., Boros, J., Madakashira, B.P., Robinson, M.L., Reneker, L.W., Mcavoy, J.W., Lovicu, F.J., 2010. Sef is a negative regulator of fiber cell differentiation in the ocular lens. Differentiation 80, 53e67. Ozaki, K., Miyazaki, S., Tanimura, S., Kohno, M., 2005. Efficient suppression of FGF-2induced ERK activation by the cooperatie interaction among mammalian sprouty isoforms. J. Cell Sci. 118, 5861e5871. Quintanar-Audelo, M., Yusoff, P., Sinniah, S., Chandramouli, S., Guy, G.R., 2011. Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology 1domain-containing protein (SPRED1), a tyrosine-protein phosphatase nonreceptor type 11 (SHP2) substrate in the Ras/extracellular signal-regulated kinase (ERK) pathway. J. Biological Chem. 286, 23102e23112. Shin, E.H.H., Basson, M.A., Robinson, M.L., Mcavoy, J.W., Lovicu, F.J., 2012. Sprouty is a negative regulator of transforming growth factor b-induced epithelial-tomesencymal transition and cataract. Mol. Med. 18, 861e873. Srinivasan, Y., Lovicu, F.J., Overbeek, P.A., 1998. Lens-specific expression of transforming growth factor b1 in transgenic mice causes anterior subcapsular cataracts. J. Clin. Investigation 101, 625e634. Tefft, J.D., Lee, M., Smith, S., Leinwand, M., Zhao, J., Bringas Jr., P., Crowe, D.L., Warburton, D., 1999. Conserved function of mSpry-2, a murine homolog of Drosophila sprouty, which negatively modulates respiratory organogenesis. Curr. Biol. 9, 219e222. Torri, S., Kusakabe, M., Yamamoto, T., Maekawa, M., Nishida, E., 2004. Sef is a spatial regulator for Ras/MAP kinase signaling. Dev. Cell. 7, 33e44. Wakioka, T., Sasaki, A., Kato, R., Shouda, T., Matsumoto, A., Miyoshi, K., Tsuneoka, M., Komiya, S., Baron, R., Yoshimura, A., 2001. Spred is a sprouty-related suppressor of Ras signaling. Nature 412, 647e651. Xie, L., Law, B.K., Chytil, A.M., Brown, K.A., Aakre, M.E., Moses, H.L., 2004. Activation of the Erk pathway is required for TGF-beta1-induced EMT in vitro. Neoplasia 603e610.
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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 b
Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia Save Sight Institute, University of Sydney, NSW, Australia
a r t i c l e i n f o
a b s t r a c t
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
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 RTKantagonists (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 TGFbinduced 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 overexpression 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.
Keywords: TGFb Lens pathology EMT RTK antagonists Spry Sef Spred
1. Introduction The establishment and maintenance of the distinctive architecture 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
* Corresponding author. Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, NSW, Australia. E-mail address: [email protected] (F.J. Lovicu). http://dx.doi.org/10.1016/j.exer.2015.01.001 0014-4835/© 2015 Elsevier Ltd. All rights reserved.
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
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deficient for Sprouty (Spry) are more sensitive to this TGFb-induced insult, leading to aberrant cell behavior, EMT and subsequent cataract (Shin et al., 2012). Sef, Spry and Spred (Sprouty-related protein) belong to families of negative-regulatory molecules reported to act on receptor tyrosine kinases (RTKs), including the FGF-Ras-ERK/MAPK pathway (Hacohen et al., 1998; Casci et al., 1999; Kramer et al., 1999; Chambers and Mason, 2000; Wakioka et al., 2001; Li et al., 2003; Ozaki et al., 2005). Spry and Sef are normally expressed in the lens and eye throughout embryogenesis and postnatal growth (Boros et al., 2006), consistent with them playing an important role during early lens and eye development (Newitt et al., 2010; Kuracha et al., 2011, 2013), as well as in other tissues and organs (Kramer et al., 1999; Tefft et al., 1999; Chambers and Mason, 2000; Boros et al., 2006; Mason et al., 2006). Although a deficiency of Sef in the lens does not appear to have an impact on lens development (see Newitt et al., 2010), when Spry1 and Spry2 are conditionally deleted from the murine lens, postnatally, the lens epithelia of these mutant mice undergo aberrant cell behavior leading to cataract (Shin et al., 2012). This deficiency of Spry in the lens was shown to impair lens epithelial intracellular signaling, including increased phosphorylation of ERK1/2, as well as nuclear localization of Smad2/3, snail and slug (downstream targets of TGFbsignaling) that lead to deregulation of E-cadherin and upregulation of alpha-smooth muscle actin (a-sma), all leading to transdifferentiation of cells into myofibroblastic cells with aberrant deposition of extracellular matrix, defining an EMT (Shin et al., 2012). More convincingly, overexpression of Spry in lens epithelia, both in vitro and in situ, was shown to effectively block the influence of TGFb, as evident by the repression of nuclear localization of phosphorylated Smads, snail and slug, as well as a block in a-sma accumulation, and maintenance of E-cadherin (Shin et al., 2012). In situ, this translated to Spry effectively preventing/ blocking TGFb-induced EMT and cataract. Based on these studies we proposed that RTK antagonists, such as Spry, may play an important role in the maintenance of the normal monolayer of lens epithelia, protecting it to some degree from the detrimental effects of deregulated TGFb signaling. Moreover, for TGFb to elicit its effects on the lens, we would expect that Spry itself would need to be downregulated, and indeed this is the case, supported by gene array studies demonstrating that Spry1 and Spry2 levels are both reduced in lens cells (FLH124) exposed to TGFb (Dawes et al., 2007). To better understand the role of the different RTK antagonists expressed in the lens, we screened other putative negative regulators of TGFb-mediated EMT, using the lens epithelial explant system. For this we examined and compared different RTK inhibitors including Sprouty1, Sprouty2 and Sef, as well as members of the Spred family, Spred1, Spred2 and Spred3; that we show here to also be expressed normally in the eye and lens cells. Using this approach, we demonstrate that all the antagonists tested can reduce the ability of TGFb to induce an EMT in lens epithelial cells. This data supports a key role for negative regulators of intracellular signaling, to protect lens cells not only from the adverse effects of compounds such as TGFb, but also to maintain lens cell integrity and hence transparency. 2. Material and methods All animal handling and operating procedures carried out in this study adhered to the ARVO statement for the use of animals in ophthalmic research, conforming to the provisions of the code of practice provided by the National Health and Medical Research Council (NHMRC, Australia), and were approved by the Animal Ethics Committee of the University of Sydney, NSW, Australia. Ocular tissues were derived from postnatal day 3 (P3), P15 and P21
albino Wistar rats. 2.1. RT-PCR Unless otherwise stated, steps utilizing commercial reagents and kits were carried out according to manufacturer instructions. TriReagent (Molecular Research Centre, OH, USA) was used to extract total RNA from the following tissues harvested from P15 rats: whole eye, whole lens, lens capsule (with adherent epithelial cells), and lens fibers. To remove DNA that may have co-purified with the RNA, each sample was incubated with DNase I (Amersham Biosciences, NJ, USA) for 30 min at 37 C. Treatment with DNase Inactivation Reagent (Life Technologies, CA, USA) was carried out to remove the enzyme and divalent cations that would otherwise be detrimental to downstream DNA synthesis. First strand cDNA was reverse transcribed from 2 mg of the prepared total RNA using Promega's Reverse Transcription System (WI, USA). Control reactions omitting AMV reverse transcriptase, were set up and later tested, confirming that genomic DNA was absent from the purified RNA template (data not shown). To investigate Spred expression, gene specific primers for coding regions of Spred1 (50 agacacagcctccctcgtta-30 , 50 -ttatgtttcccaccacagca-30 ), Spred2 (50 cgggtgagctgtatgtggt-30 , 50 -atacaaggggccaggaaaga-30 ) and Spred3 (50 -cggatgagttccagaagagc-30 , 50 -gatcgccggtagtcctcata-30 ) were incorporated into PCR reactions containing 5 ml cDNA, 1.7 mM MgCl2, 1 mM dNTPs, 1x NH4 reaction buffer and 0.8 U BioTaq Red™ (Bioline, London, UK). Reactions were denatured at 94 C for 3 min, amplified through 32 cycles (94 C for 30 s, 58 C for 30 s, 72 C for 1 min) and allowed a final extension at 72 C for 2 min. The end PCR products were analyzed by agarose gel electrophoresis. 2.2. SDS-PAGE and western blotting Lens epithelial cells and fiber cell samples were collected from P15 rats and homogenized in cold lysis buffer containing 2.5 mM EDTA, 25 mM TriseHCl (pH 7.5), 0.375 M NaCl, 1% IGEPAL, 1.5 mM sodium orthovanadate and a protease inhibitor cocktail (complete, Mini; Roche, Basel, Switzerland). To facilitate lysis, the homogenized samples were rotated for 2 h at 4 C. Protein quantification using the Micro-BCA protein assay kit (Pierce, IL, USA) enabled size separation of 20 mg of total protein through a 10% SDS-PAGE matrix. Prior to gel loading, protein lysates were mixed in a 1:1 ratio with Laemmli sample buffer (BioRad, CA, USA) containing 5% (v/v) 2mercaptoethanol as a denaturant. Once separated, proteins were transferred from the gel matrix to a PVDF membrane (Millipore). The membrane was incubated (1 h at RT) with a blocking solution of 5% (w/v) skim milk powder in TBS-T (0.1% (v/v) Tween 20 in trisbuffered saline (TBS) and then probed overnight with polyclonal antibodies generated from GST fusion proteins of human Spred1, 2 or 3 (diluted 1:1000 in blocking solution). Antibody specificity was verified by Western blot analysis of transfected HEK-293T cell lysates (see King et al., 2005). Unbound antibody was removed with multiple TBS-T washes, before a 2 h incubation with goat antirabbit HRP-conjugated IgG (diluted 1:5000 in TBS-T; Cell Signaling Technologies, MA, USA). Again, unbound antibody was removed with TBS-T washes, this time followed by application of Immobilin Western Chemiluminescent HRP substrate (Millipore). Hyperfilm™ (Amersham Biosciences) was used to capture the chemiluminescence, which was visualized using standard autoradiography techniques. 2.3. Immunofluorescent labeling of explants and whole-mounts Lens epithelial explants were prepared as described previously (Lovicu and McAvoy, 2008), and were cultured in Medium 199 with
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Earle's salts (M199; Trace Scientific, NSW, Australia), supplemented with 0.1% bovine serum albumin (BSA; Sigma, NSW, Australia), 0.1 mg/ml L-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml Amphostat (all from Trace Scientific, Australia) at 37 C in 5% CO2. Explants were cultured with 100 pg/ml TGFb-2 (R&D Systems, MN, USA) for 3 days. Prior to immunoflourescent labeling, explants were viewed with phase-contrast microscopy (CK2, Olympus, Japan) and photographed (DFC-280, Leica, Germany). Lens epithelial wholemounts or explants were fixed with 100% methanol for 45 s and rinsed in succession with PBS. Following rinses (3 5 min) with 0.1% (w/v) BSA in PBS (PBS/BSA), explants were incubated with a 10% normal goat serum (NGS) blocking agent (1 h at RT). Excess blocking buffer was removed and anti-Spred antibodies (described above) or mouse-anti-a-sma (1:100 dilution in 1.5% NGS; Sigma, NSW, Australia) was applied to the explants for an overnight incubation at 4 C. Excess primary antibody was removed with rinses in PBS/BSA (3 5 min), prior to the application of secondary antibody. Antimouse- or anti-rabbit-Alexa 488 or Alexa-594 (all antibodies diluted 1:1000 in PBS/BSA; Invitrogen, USA) were applied to explants and left for 2 h at room temperature. Cell nuclei were visualized with Hoechst dye (6 mg/ml; Calbiochem, USA) for 5 min. Following 3 5 min washes in PBS/BSA, the explants were mounted with 10% PBS (v/v) in glycerol and immunofluorescent labeling was visualized and imaged using confocal microscopy (Zeiss LSM 700). 2.4. DNA transfection of lens epithelial explants
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buffered formalin (NBF) for 20 min, and rinsed with 70% ethanol (3 5 min). For lectin staining of lens cell membranes, explants were rehydrated in 0.1% (w/v) BSA in PBS (PBS/BSA; 3 5 min washes) at room temperature. The explants were then permeabilized in 0.05% Tween-20 in PBS/BSA (3 5 min), and rinsed in PBS/BSA (2 5 min). After wiping off excess fluid, lectin-TRITC (1:100 dilution in PBS/BSA; Sigma, NSW, Australia) was applied directly onto the explant for 1 h at room temperature. Explants were then rinsed with PBS/BSA (3 5 min) prior to nuclear labeling with Hoechst dye (Calbiochem) for 5 min. Explants were mounted in 10% PBS (v/v) in glycerol and visualized using confocal microscopy (Zeiss LSM 700). 3. Results 3.1. Spred expression in the eye Using P15 Wistar rat ocular tissues, RT-PCR demonstrated that all Spreds; Spred1, Spred2 and Spred3, were expressed in the eye (Fig. 1A). This ocular expression, at least in part, was due to the expression of Spred in the intact lens, with stronger expression demonstrated in the lens epithelium, compared to the lens fiber cells. The lens expression translated to the presence of the respective Spred proteins in lysates of corresponding lens epithelia (Fig. 1B). In P3 lens/eye tissue sections we see strong immunofluorescent labeling for Spreds, for example Spred-1 in epithelia of the cornea, ciliary body and lens, as well as in the lens fibers (Fig. 2). Strong labeling was seen at the epithelial to fiber cell interface
The construction of DNA plasmids of interest (pLXSG, pLXSGSprouty1, pLXSG-Sprouty2, pLXSG-Sef, pLXSG-Spred1, pLXSGSpred2 and pLXSG-Spred3) were confirmed by sequencing and extracted and purified according to manufacturer's instructions (Astral scientific, Australia). Using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA), 2 mg of DNA plasmid encoding for the antagonist of interest was obtained. Lipofectamine 2000 (Invitrogen, USA) was diluted in unsupplemented M199 media (1:10 dilution), and mixed with 2 mg of DNA and kept at room temperature for 20 min to form the DNA-Lipofectamine complexes. The DNA-Lipofectamine complexes were added to culture dishes containing epithelial explants (described above) and incubated at 37 C, 5% CO2 for 48 h. To confirm the successful transfection of the explants, the expression of enhanced-GFP was visualized using an inverted fluorescent microscope (Leica DMLB, Germany). Culture media was replaced with freshly supplemented M199 medium, and explants were then treated with 100 pg/ml TGFb-2 for up to 3 days. 2.5. Quantification of TGFb-induced EMT The successfully transfected lens epithelial cells expressing EGFP were photographed after 3 days of culture. Cell counts were carried out in a blind test to eliminate bias. An epithelial cell was defined as being cuboidal in shape, similar to epithelial cells prior to the addition of TGFb. A cell that elongated and was fibrotic in appearance, extending cellular processes beyond the normal boundaries of an epithelial cell was considered to have undergone an EMT. The percentage of labeled cells undergoing an EMT was determined and statistical significance was inferred using a paired two tailed t-test. 2.6. Lectin staining of transfected lens epithelial explants Transfected lens epithelial explants were fixed with 10% neutral
Fig. 1. (A) RT-PCR was used to detect Spred expression in postnatal day 15 rat eye (lane 1), lens (lane 2), lens epithelium (lane 3) and lens fibers (lane 4). A negative control, no DNA sample (lane 5) and positive control, Spred plasmid (lane 6) and size markers (lane 7) are also shown. Spred1, Spred2 and Spred3 are expressed in whole eye, with expression of Spred1-3 in lens epithelia and Spred1 and Spred2 in lens fibers. (B) Western blotting also demonstrated Spred1-3 protein in lens epithelial cell lysates. atubulin was used as a loading control.
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Fig. 2. Representative immunofluorescent labeling for Spred (Spred-1) in ocular sections of neonatal (P3) rats (C,D), counterstained with Hoechst dye for nuclei (A,B). Spred-1 is strongly expressed in both lens epithelial cells (le) and fiber cells (lf), with strong labeling at their interface (C,D, arrows). Strong labeling was also observed in corneal epithelia (ce) as well as developing ciliary body and iris. Whole mounts of lens epithelia also demonstrated membrane labeling for Spred-1 in fiber cells (E, green) and some cytoplasmic labeling in epithelial cells (F). Note that in whole mounts of lens epithelium, patches of adherent apical tips of fibers are frequently retained and can be visualised in optical sections. Scale Bar, 20 mm.
(Figure AeD, arrows), which was also highlighted in lens epithelial whole-mounts, demonstrating membrane labeling at the apical tips of fiber cells (Fig. 2E), as well as cytoplasmic labeling in many of the epithelial cells (Fig. 2F). 3.2. TGFb-induced epithelial-to-mesenchymal transition (EMT) Lens epithelial explants treated with or without TGFb were collected after 3 days of culture, and any morphological changes induced in the epithelial cells were initially observed using phasecontrast microscopy. Explants were immunolabeled for a-sma. The presence of TGFb induced a distinctive transition in cell morphology over the 3 days of culture, with the lens epithelial cells undergoing an EMT characterized by cell elongation into spindlelike cells (Fig. 3B, arrows). There was also evidence of cell loss due to apoptosis (Fig. 3B, asterisk; see Maruno et al., 2002) with distinct regions devoid of cells, exposing the lens capsule that displayed capsular wrinkling (Fig. 3B, arrowheads). Explants cultured with TGFb demonstrated strong labeling for a-sma in distinct stress fibers, consistent with a mesenchymal phenotype (Fig. 3D). In contrast, control explants showed no notable cellular changes over the culture period with the monolayer of epithelial cells retaining their cuboidal morphology (Fig. 3A). Consistent with this, the control cells expressed basal levels of amorphous a-sma labeling (Fig. 3C). 3.3. RTK-antagonists block TGFb-induced EMT To examine the efficiency of growth factor antagonists in preventing TGFb-induced EMT, cells in lens epithelial explants were transfected with a panel of different antagonists normally expressed in the lens (Spry1, Spry2, Sef, Spred1, Spred2 and Spred3). Successful transfection was evident by the expression of
reporter protein (EGFP), indicative of plasmid uptake by the cells (Fig. 4). Live transfected cells were viewed and imaged, and cell counts were used to quantify the percentage of cells undergoing fibrotic cell changes induced by TGFb. Using lectin membrane labeling we could observe the transfected cells in the context of the surrounding non-transfected cells within the explant. Fig. 4 shows representative results for the Spry2 and Spred3 transfection studies as examples of the effects of the different treatments. When explants were transfected with pLXSG alone (reporter plasmid) and cultured without TGFb for 3 days (Fig. 4A, E), the EGFP-expressing (labeled) cells maintained their irregular cuboidal shape, indicating that EGFP expression has no direct influence on lens epithelial cell integrity. However, when these same cells were treated with TGFb (Fig. 3B, F), the pLXSG transfected cells elongated by 3 days, typical of a TGFb-induced fibrotic/EMT response. This was consistent with the surrounding non-EGFP expressing cells (Fig. 4F), demonstrating that EGFP expression alone is not sufficient to inhibit TGFb-induced EMT. On average, 61% of the cells in explants that were transfected with pLXSG alone and treated with TGFb underwent fibrotic-like cellular changes (Fig. 5). With the addition of TGFb, the morphology of epithelial cells that were not transfected (and thus not expressing EGFP) transitioned normally into a mesenchymal phenotype and exhibited cell loss consistent with a TGFb-induced EMT response (Fig. 4FeH). Given the relatively low transfection efficiency of our primary explants, we could not effectively assay or quantify for changes in any molecular markers under these conditions; however, when we doublelabeled for EGFP-expressing transfected cells (for example, overexpressing Spry2) with a-sma, we noticed a reduced staining for asma in these cells that failed to undergo an EMT in response to TGFb. Compared to non-transfected neighboring cells displaying a more prominent mesenchymal phenotype, a-sma was weaker and mostly diffuse (not localized to stress fibers) in transfected cells
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Fig. 3. TGF-b induced EMT after 3 days. (A) Phase contrast microscopy of explants cultured without TGFb show the tightly packed monolayer of cuboidal cells. (B) When explants were cultured with 100 pg/ml of TGFb for 3 days, cell elongation (arrows), exposed regions of lens capsule (asterisk) and capsular wrinkling (arrowheads) are evident. (C) Immunoflourescent labeling showed that explants cultured without TGFb exhibited low levels of amorphous a-sma labeling in the cytoplasm (green). Hoechst nuclear counterstaining (red) demonstrates rounded nuclei consistent with an epithelial phenotype. (D) Explants cultured with TGFb exhibit distinct a-sma immunoreactivity in stress fibers, with nuclear staining revealing more elongate and irregular shaped nuclei, consistent with a mesenchymal phenotype.
that failed to undergo an EMT in response to TGFb (see Fig. 6). The overexpression of RTK-antagonists was not sufficient to initiate cellular changes associated with EMT without the presence of TGFb. Explants retained their tightly packed, cuboidal monolayer under these conditions (data not shown). When cells were transfected with Spry1 and Spry2 there was a significant decrease in the percentage of cells elongating and undergoing a TGFb-induced EMT, compared to control explants (p < 0.001). A 64% reduction in labeled cells demonstrating fibrotic changes resulted when epithelial cells were transfected with pLXSG-Spry1 and exposed to TGFb (Fig. 5). Similarly, when transfected with pLXSG-Spry2, 71% of the labeled lens epithelial cells did not undergo TGFb-induced EMT. The overexpression of Sef (pLXSG-Sef) led also to a significant inhibition of TGFb-induced EMT, with 76% (p < 0.001) of transfected/ labeled cells remaining cuboidal when cultured with TGFb. The Spred family of RTK-antagonists, of which we demonstrate here to be also present in the lens, appear to be the most potent inhibitors of TGFb-induced EMT. Transfection of lens epithelial cells with pLXSG-Spred1 significantly decreased TGFb-induced fibrotic changes by 92% compared to control explants (p < 0.001). The overexpression of Spred2 (pLXSG-Spred2) resulted in an 82% reduction, whereas with Spred3, a 91% reduction; a significant
inhibition of TGFb-induced EMT in all cases (p < 0.001). In summary, the overexpression of RTK-antagonists in the presence of TGFb leads to a significant reduction of the TGFbassociated cellular changes; however, it does not lead to a complete block in the TGFb-induced EMT. Most cells in explants that were transfected with RTK-antagonists and treated with TGFb retained the typical cuboidal epithelial shape (Fig. 4G, H), while others (albeit a smaller percentage) were still capable of undergoing an EMT. 4. Discussion Transforming growth factor b (TGFb) induces an epithelial-tomesenchymal transition (EMT) that results in lens opacities characteristic of those found in some forms of human cataract (see De Iongh et al., 2005). Accordingly, the actions of TGFb are tightly regulated in normal lens cell biology. One mode of regulation is proposed to be via intracellular antagonists reported to regulate RTK-induced intracellular signaling pathways. Antagonists of the MAPK/ERK signaling pathway such as Spry and Sef have been shown to be strongly expressed in the lens epithelium (Boros et al., 2006; Shin et al., 2012) and we show here that other antagonists
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Fig. 4. DNA transfection with different RTK antagonists inhibits TGFb induced EMT. Transfected cells express EGFP (green). Without TGFb, pLXSG-expressing cells retained a typical irregular cuboidal shape (A), consistent with the surrounding cells visualised by lectin staining (E). When cultured with TGFb for 3 days, pLXSG expressing cells and surrounding cells underwent EMT and elongated (B, F). Cells transfected with pLXSG-Spry (C, G) and pLXSG-Spred (D, H), retained a cuboidal shape, whereas surrounding cells elongated and underwent an EMT as evident by cell loss and areas of bare capsule.
Fig. 5. Comparison of the percentage of TGF-b-induced EMT in lens epithelia cells overexpressing the RTK-antagonists of interest (Spry1, Spry2, Sef, Spred-1, Spred-2 and Spred-3) or only the reporter gene (pLXSG). A significant reduction of TGFb-induced change in morphology is evident in the presence of the antagonists (n ¼ 20 for each RTK-antagonist). *P < 0.001.
including Spred1, Spred2 and Spred3 are also expressed in the lens. The current investigation provides a first screen of these selective MAPK/ERK1/2 antagonists, Spry, Sef and Spred to determine how effective they are at blocking TGFb-induced EMT. All antagonists examined were shown to have the ability to block TGFb-induced
EMT, with Spred1 in particular found to be the most potent antagonist in this system. This may be attributed to the larger Nterminal Enabled/VASP homology 1 (EVH1) domain of Spred1 (McClatchey and Cichowski, 2012), which may confer an increased ability to inhibit, compared to other Spreds, for example (King et al.,
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Fig. 6. TGFb-induced EMT after 3 days. Immunoflourescent labeling showed that cells in lens explants cultured with TGFb exhibited distinct a-sma immunoreactivity, notably in stress fibers (red, arrows; C,D), consistent with a mesenchymal phenotype; however, transfected cells co-labeled for EGFP (B,D; overexpressing Spry2 in this instance) exhibited lower levels of a more amorphous a-sma-labeling in the cytoplasm (arrowhead). Hoechst dye counterstaining (blue; A,D) highlights the cell nuclei. Scale Bar, 50 mm.
2005). TGFb has a potent cataractogenic effect on lens epithelial cells, disrupting the monolayer of cuboidal cells and cellular integrity, resulting in a morphological transition into elongate myofibroblastic/fibroblastic cells. These cells also express a-sma in response to TGFb, a hallmark of EMT. Whilst were able to demonstrate that transfected cells failing to undergo an EMT in response to TGFb had reduced levels of a-sma, we would require a greater cell transfection efficiency to quantify this. We did however note that a-sma in these cells was not distributed in stress fibers, typical of myofibroblastic cells as shown in neighboring non-transfected cells responding to TGFb. We must be mindful that the RTK antagonists overexpressed may be uncoupling the EMT process by only targeting select signaling pathways impacting on cell morphology, and not all signaling (downstream of TGFb) leading to an EMT that may involve a-sma accumulation. Although all the antagonists tested were shown to potentially block TGFb-induced cellular responses in this system, the presence of both cuboidal epithelial cells, that resisted undergoing an EMT, as well as cells that still retained the ability to undergo morphological changes typical of an EMT, highlights the different efficiency of the individual antagonists. This variability in effectiveness of each antagonist may also depend upon the level of antagonist expression within specific cells. This is a variable of our in vitro system that we could not control. Nevertheless, overexpression studies in situ provide further validation of the antagonistic effects of the RTKinhibitors on the TGFb-induced cellular responses. For example, some of our recent studies have shown that overexpression of Spry
in transgenic mice can ameliorate anterior subcapsular cataract (ASC) induced by TGFb overexpression (Shin et al., 2012). Consistent with this, knock-out studies of Spry proteins in the murine lens result in ASC plaque formation and increases in TGFb signaling, including induced nuclear localization of Smad2/3, snail and slug, similar to that found in mice overexpressing TGFb (Shin et al., 2012). Given that Spry proteins are required for lens epithelial integrity and have a profound effect on TGFb-induced cataract in situ, as well as the ability to significantly hamper the TGFb-inducedEMT when overexpressed through the transfection of lens epithelial explants, it is clear that the Spry family of proteins have a crucial role in the regulation of the TGFb-induced EMT. Sef has also been shown to be strongly expressed in the lens epithelium during embryogenesis and postnatally (Boros et al., 2006). The present study is the first to examine the role of Sef protein in the context of TGFb-induced EMT of the lens. It was shown that the overexpression of Sef is more effective than both Spry1 and Spry2 at blocking the TGFb-induced cellular responses within explants. Given that Sef has been proposed to block FGF signaling in lens (Newitt et al., 2010), as well as TGFb-induced EMT in our system, it would be interesting to further examine the role FGF plays in TGFb-induced EMT. FGF has previously been shown to induce ERK1/2 signaling (Lovicu and McAvoy, 2001), a primary target of these antagonists (Hanafusa et al., 2002; Torri et al., 2004; Quintanar-Audelo et al., 2011). In other systems, such as rat mesangial cells (Jiang et al., 2010) and mouse cortical tubule cells (Xie et al., 2004), TGFb has been shown to signal through Smadindependent signaling such as the MAPK/ERK pathway during
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EMT. The putative cross-talk between the MAPK/ERK and canonical TGFb signal transduction pathways identifies a possible mechanism for these growth factor antagonists to inhibit TGFb-induced EMT. As previous studies have shown Spry, Sef and Spred to antagonize the MAPK/ERK signaling pathway (Hanafusa et al., 2002; Torri et al., 2004; Quintanar-Audelo et al., 2011), it is likely that these specific RTK-antagonists are exerting their actions on MAPK/ERK signal transduction to block TGFb-induced lens EMT. Further studies to determine how these RTK-antagonistic molecules slow or block the progression of subcapsular plaque formation, and the role for ERK1/ 2, will provide greater insights into the mechanism of TGFbmediated cataract formation in situ. In summary, it is clear that these antagonists impede the fibrotic cellular changes associated with TGFb-induced EMT in situ (for Spry) and now within lens epithelial explants. Spry, Sef and Spred may therefore serve as putative therapeutic agents for the prevention of EMT, and thus cataractogenesis. Acknowledgments The authors would like to thank A/Prof Christopher Hovens (University of Melbourne, Vic, Australia) for providing the Spred antibodies. The work was funded by the NIH (R01 EY03177), USA, the National Health and Medical Research Council, and the Sydney Foundation for Medical Research, Australia. References Boros, J., Newitt, P., Wang, Q., Mcavoy, J.W., Lovicu, F.J., 2006. Sef and sprouty expression in the developing ocular lens: implications for regulating lens cell proliferation and differentiation. Seminars Cell Dev. Biol. 17, 741e752. Casci, T., Vinos, J., Freeman, M., 1999. Sprouty, an intracellular inhibitor of Ras signaling. Cell 96, 655e665. Chambers, D., Mason, I., 2000. Expression of sprouty2 during early development of the chick embryo is coincidet with known sites of FGF signalling. Mech. Dev. 91, 361e364. Dawes, L.J., Elliot, R.M., Reddan, J.R., Wormstone, Y.M., Wormstone, I.M., 2007. Oligonucleotide microarray analysis of human lens epithelial cells: TGFb regulated gene expression. Mol. Vis. 1181e1197. De Iongh, R.U., Bellusci, S., Lovicu, F.J., Mcavoy, J.W., 2005. Transforming growth factor-b induced epithelial-mesenchymal transition in the lens: a model for cataract formation. Cells Tissues Organs 179, 43e55. Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y., Krasnow, M.A., 1998. Sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the Drosophila airways. Cell 92, 253e256. Hanafusa, H., Torri, S., Yasunaga, T., Nishida, E., 2002. Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signaling pathway. Nat. Cell. Biol. 4, 850e858. Jiang, W., Zhang, Y., Wu, H., Zhang, X., Gan, H., Sun, J., Chen, Q., Guo, M., Zhang, Z., 2010. Role of cross-talk between the Smad2 and MAPK pathways in TGF-b1induced collagen IV expression in mesangial cells. Int. J. Mol. Med. 26, 571e576. King, J.A.J., Straffon, A.F.L., D'Abaco, G.M., Poon, C.L.C., Stacey, T.T.I., Smith, C.M., Buchert, M., Corcoran, N.M., Hall, N.E., Callus, B.A., Sarcevic, B., Martin, D., Lock, P., Hovens, C.M., 2005. Distinct requirements for the sprouty domain for
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