FGF-2 Differentially Regulates Lens Epithelial Cell Behaviour during TGF-β-Induced EMT

Fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-β) can regulate and/or dysregulate lens epithelial cell (LEC) behaviour, including proliferation, fibre differentiation, and epithelial–mesenchymal transition (EMT). Earlier studies have investigated the crosstalk between FGF and TGF-β in dictating lens cell fate, that appears to be dose dependent. Here, we tested the hypothesis that a fibre-differentiating dose of FGF differentially regulates the behaviour of lens epithelial cells undergoing TGF-β-induced EMT. Postnatal 21-day-old rat lens epithelial explants were treated with a fibre-differentiating dose of FGF-2 (200 ng/mL) and/or TGF-β2 (50 pg/mL) over a 7-day culture period. We compared central LECs (CLECs) and peripheral LECs (PLECs) using immunolabelling for changes in markers for EMT (α-SMA), lens fibre differentiation (β-crystallin), epithelial cell adhesion (β-catenin), and the cytoskeleton (alpha-tropomyosin), as well as Smad2/3- and MAPK/ERK1/2-signalling. Lens epithelial explants cotreated with FGF-2 and TGF-β2 exhibited a differential response, with CLECs undergoing EMT while PLECs favoured more of a lens fibre differentiation response, compared to the TGF-β-only-treated explants where all cells in the explants underwent EMT. The CLECs cotreated with FGF and TGF-β immunolabelled for α-SMA, with minimal β-crystallin, whereas the PLECs demonstrated strong β-crystallin reactivity and little α-SMA. Interestingly, compared to the TGF-β-only-treated explants, α-SMA was significantly decreased in the CLECs cotreated with FGF/TGF-β. Smad-dependent and independent signalling was increased in the FGF-2/TGF-β2 co-treated CLECs, that had a heightened number of cells with nuclear localisation of Smad2/3 compared to the PLECs, that in contrast had more pronounced ERK1/2-signalling over Smad2/3 activation. The current study has confirmed that FGF-2 is influential in differentially regulating the behaviour of LECs during TGF-β-induced EMT, leading to a heterogenous cell population, typical of that observed in the development of post-surgical, posterior capsular opacification (PCO). This highlights the cooperative relationship between FGF and TGF-β leading to lens pathology, providing a different perspective when considering preventative measures for controlling PCO.

In the current study, we characterized the influence that FGF has on TGF-β-induced cell behaviour in rat lens explants to best model the conditions needed to promote a heterogenous cell population typical of fibrotic cataracts as seen in situ. We demonstrate that a high fibre-differentiating dose of FGF is protective of TGF-β-induced EMT in peripheral lens epithelia; however, this is not evident in central lens epithelia induced by TGF-β. This emulates the spatial phenotypic response of lens cells seen in human PCO and may serve as a model to better understand the mechanisms leading to this post-surgical pathology.

Animals and Tissue Culture
All procedures conducted abided by the Australian Code for animal care and usage for scientific purposes and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals for Ophthalmic and Vision biomedical research (USA). The experiments were approved by the Animal Ethics Committee of The University of Sydney, NSW, Australia (AEC# 2021/1913). Wistar rats (rattus norvegicus) at 21-days of age (P21 ± 1 day) were humanely euthanized with CO 2 followed by cervical dislocation.

Lens Epithelial Explants
All collected primary rat ocular tissues were kept in medium 199 with Earle's Salts (M199) (11825015, Gibco TM , Thermo Fisher Scientific, Sydney, NSW, Australia) in 35 mm Nunc™ culture dishes (NUN150460, Thermo Fisher Scientific). The media was supplemented with 2.5 µg/mL Amphotericin B (15290-018, Gibco TM , Thermo Fisher Scientific), 0.1% bovine serum albumin (BSA) (9048-46-8, Sigma-Aldrich Corp., St. Louis, MO, USA), 0.1 µg/mL L-glutamine (200 mM) (25030081, Gibco TM , Life Technologies, Carlsbad, CA, USA), and penicillin (100 IU/mL)/streptomycin (100 µg/mL) (15140-122, Gibco TM , Life Technologies). The collected eyes were placed under a dissecting microscope to remove the lenses. The posterior capsule of the lens was torn using fine forceps and the remaining intact anterior capsule containing a sheet of lens epithelial cells (LECs) was pinned to the base of the culture dish using the gentle pressure of the forceps, as previously described [5]. Explants were maintained in a humidified incubator (37 • C, 5% CO 2 ). Different doses of recombinant human TGF-β2 (302-B2-002, R&D systems, Minneapolis, MN, USA) were used to induce EMT in the lens explants, as previously described [39]. A lower dose of TGF-β2 (50 pg/mL) gave a more regulated EMT response over 7 days, while a higher dose (200 pg/mL) was used to induce a more rapid EMT response in the lens explants over this same time period. To determine the impact of FGF-2 on TGF-β2-induced lens EMT, we cotreated TGF-β2-treated LECs with either a low proliferating dose of recombinant human FGF-2 (5 ng/mL: 233-FB, R&D systems) or a high fibre-differentiating dose of FGF-2 (200 ng/mL) [10,50]. Control explants had no growth factors added to the media.

Assessment of Cell Morphology and Immunofluorescence
Cultured LEC explants were monitored and photographed daily over 7 days using phase contrast microscopy (Leica FireCam imaging, Leica Microsystems, Version 1.5, 2007). To examine the extent of how transdifferentiated cells modulated the underlying lens capsule, some treated explants were rinsed with filtered Milli-Q H 2 O to debride all cells from the explant to completely expose the underlying lens capsule. Phase contrast images were captured before and after rinsing.

SDS-Page and Western Blotting
Cultured lens epithelial explants at set time points were rinsed in cold PBS. The central and peripheral regions of the explants were isolated using a scalpel blade to delineate each region. A central square of tissue, no more than a third of the explant diameter (central LECs, CLECs), and the remaining surrounding peripheral LECs (PLECs) were isolated separately. CLEC and PLEC protein was harvested, pooled into allocated Eppendorf tubes, and lysed with cold radioimmunoprecipitation assay (RIPA) lysis buffer containing 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% Sodium dodecyl sulphate (SDS), 1 mM sodium orthovanadate, 1 mM NaF, 50 mM Tris-HCl (pH 7.5), 0.1% Triton X-100, phosphatase (PhosSTOP TM ), and protease (cOmplete TM ) EASYpacks inhibitor tablets (04906837001 and 05892970001; Roche Applied Science, Basel, Switzerland). LECs were homogenized and centrifuged for 10 min at 4 • C (14,400× g) for lysate/supernatant separation. Quantification of the total lens protein of each supernatant sample was conducted using a Pierce TM Micro bicinchoninic acid (BCA) protein assay reagent kit (23235; Thermo Fisher Scientific).
Following immunolabeling, PVDF membranes were stripped for 10 min in stripping buffer (10% SDS, 0.5 M Tris HCl pH 6.8, Milli-Q H 2 O, and 0.8% 2-mercaptoethanol) with gentle agitation. The membranes were then washed in TBST (3 × 5 min) and re-blocked in blocking buffer/TBST for 1 h. Following blocking, the membranes were probed for loading control GAPDH (1:2000, 1 h) and incubated with an HRP-conjugated horse antimouse secondary antibody for 1 h prior to chemiluminescent signalling analysis. Protein densitometry was carried out using Bio-Rad ImageLab software (Version 6.1.0, 2019).

Statistical Analysis
For each experimental analysis, three independent experiments were carried out. For every experiment, a minimum of three individual replicates (n = 3) per treatment group (different treatment of explants) were used. For Western blotting, each group contained up to eight explants to isolate central and peripheral lens cells that were randomly obtained from different P21 rats. For measuring changes in protein expression, we used densitometry to calculate the selected protein intensity relative to the loading control (GAPDH).
For Western blot experiments examining differences in Smad-dependent (Smad2/3) and Smad-independent (MAPK/ERK1/2) activity, phosphorylated protein expression was calculated using the following ratio: relative phosphorylated density per total protein.
Prior to the use of one-way analysis of variance (ANOVA), several assumptions were tested and confirmed; we assumed equal standard deviation (SD) and residuals appeared normally distributed. Based on these confirmed assumptions, we compared the differences among the means of all treatment groups using one-way ANOVA, followed by Tukey's multiple comparisons post-hoc test. All data acquired were plotted appropriately using GraphPad Prism software version 9.0 (GraphPad Software Inc., San Diego, CA, USA).
To quantify the spatial differences in t-Smad2/3 immunoreactivity, six separate images of central and peripheral regions were captured per explant across three randomized explants per treatment group, over three individual experiments. Nuclear and cytoplasmic localisation of t-Smad2/3 was manually counted using ImageJ's Cell Counter plugin. The mean percentage of t-Smad2/3 nuclear and cytoplasmic fluorescence was calculated and statistically analysed using GraphPad Prism.
Tabled data were represented as the standard error of the mean (±SEM) and probability values, where p < 0.05 was considered statistically significant.

FGF-2 Promotes a Spatially Dependent TGF-β2-Induced EMT Response in Lens Epithelial Explants
We examined the efficacy of different doses of FGF-2 in modulating the effect of TGF-β2 on lens epithelial cells induced to undergo EMT. Using phase contrast microscopy, control LECs without FGF-2 or TGF-β2 treatment ( Figure 1A,E), as well as explants treated with only a low dose of FGF-2, demonstrated no significant morphological changes and retained their epithelial phenotype over the culture period. When the lens epithelial explants were treated with a low dose of TGF-β2 (50 pg/mL), this promoted an EMT response across the entire explant, similar to a higher dose (200 pg/mL) of TGF-β2, albeit at a slower rate, consistent with previous studies [42].
With different dose combinations of FGF-2 and TGF-β2, most cells in the explants underwent a uniform EMT response over 5 days, with the exception of cells in the explants cotreated with a relatively high dose of FGF-2 (200 ng/mL) and the lower dose of TGF-β2 (Figure 1), where we observed a differential response between CLECs and PLECs (Table 1, Supplementary Figure S1).
The cells in the lens epithelial explants treated with the high a fibre-differentiating dose of FGF-2 elongated over 5 days ( Figure 1B,F), compared to the control LEC explants (no growth factor treatment; Figure 1A,E). This FGF-induced fibre differentiation response was more pronounced in PLECs compared to CLECs ( Figure 1B,F). LECs in explants treated with a low dose of TGF-β2 ( Figure 1C,G) displayed prominent EMT by day 5, with the LECs losing their uniform packing and adhesion as they transdifferentiated into myofibroblastic cells. TGF-β2 treatment also led to increased cellular blebbing (refractile bodies) and apoptotic cell loss, evident by areas of bare lens capsule that displayed prominent signs of capsular modification in the form of wrinkling throughout the explant. When the explants were cotreated with TGF-β2 and FGF-2, CLECs underwent similar morphological transformations by day 5 ( Figure 1D) to that seen with TGF-β2-treatment alone ( Figure 1C,G). In contrast, PLECs in the explants cotreated with FGF-2/TGF-β2 showed no evidence of EMT ( Figure 1H), instead demonstrating morphological changes more consistent with that observed in the explants treated with FGF-2 alone ( Figure 1B,F).
(no growth factor treatment; Figure 1A, E). This FGF-induced fibre differentiation response was more pronounced in PLECs compared to CLECs ( Figure 1B, F). LECs in explants treated with a low dose of TGF-β2 ( Figure 1C, G) displayed prominent EMT by day 5, with the LECs losing their uniform packing and adhesion as they transdifferentiated into myofibroblastic cells. TGF-β2 treatment also led to increased cellular blebbing (refractile bodies) and apoptotic cell loss, evident by areas of bare lens capsule that displayed prominent signs of capsular modification in the form of wrinkling throughout the explant. When the explants were cotreated with TGF-β2 and FGF-2, CLECs underwent similar morphological transformations by day 5 ( Figure 1D) to that seen with TGF-β2-treatment alone ( Figure 1C, G). In contrast, PLECs in the explants cotreated with FGF-2/TGF-β2 showed no evidence of EMT ( Figure 1H), instead demonstrating morphological changes more consistent with that observed in the explants treated with FGF-2 alone ( Figure 1B, F).    S3B, E) explants. In the TGF-β2-treated explants, increased capsular modulation was apparent with wrinkling and folds in the central explant regions (Supplementary Figure  S3C) and in the peripheral regions (Supplementary Figure S3F). Consistent with the differential cell response in the central and peripheral regions of the F/t-cotreated explants (Figure 2A, A1, A3), the explants exhibited capsular modulation only in the central explant region ( Figure 2A2), with no capsular wrinkling observed in the peripheral region ( Figure  2A4). We observed that with ongoing culture (up to 7 days), regardless of the explant region or treatment, all of the cells exposed to TGF-β2 (200 pg/mL) are lost by 7 days (Supplementary Figure S4A); however, in the cotreated explants (F/t), with continual supplementation of the media with FGF-2 (200 ng/mL) after day 3 of culture, this promoted cell survival, whereby we continue to observe many myofibroblastic cells in the central region of the explants (Supplementary Figure S4B) and, similarly, relatively normal lens cells at the periphery of the explants are also maintained (Supplementary Figure S4C).

Immunofluorescent Labelling
We used immunofluorescence to characterise the different cell types in explants treated with TGF-β2 and/or FGF-2 over 3 days, labeling for lens fibre differentiation markers, β-crystallin, and alpha-tropomyosin (α/9d), as well as the EMT marker, α-SMA (Figure 3). Control LECs throughout the explant exhibited no reactivity for β-crystallin and/or α-SMA after 3 days of culture ( Figure 3A), labelling only for α/9d ( Figure 3B). FGF-2treated LECs exhibited strong reactivity for β-crystallin throughout the explant (Figure We observed that with ongoing culture (up to 7 days), regardless of the explant region or treatment, all of the cells exposed to TGF-β2 (200 pg/mL) are lost by 7 days (Supplementary Figure S4A); however, in the cotreated explants (F/t), with continual supplementation of the media with FGF-2 (200 ng/mL) after day 3 of culture, this promoted cell survival, whereby we continue to observe many myofibroblastic cells in the central region of the explants (Supplementary Figure S4B) and, similarly, relatively normal lens cells at the periphery of the explants are also maintained (Supplementary Figure S4C).

Immunofluorescent Labelling
We used immunofluorescence to characterise the different cell types in explants treated with TGF-β2 and/or FGF-2 over 3 days, labeling for lens fibre differentiation markers, β-crystallin, and alpha-tropomyosin (α/9d), as well as the EMT marker, α-SMA (Figure 3). Isotype controls for all of the antibodies show little to no specific labelling. Control LECs throughout the explant exhibited no reactivity for β-crystallin and/or α-SMA after 3 days of culture ( Figure 3A), labelling only for α/9d ( Figure 3B). FGF-2-treated LECs exhibited strong reactivity for β-crystallin throughout the explant ( Figure 3C,I), with stronger labelling in PLECs ( Figure 3I). Treatment with FGF-2 did not promote α-SMA reactivity in any cultured lens epithelia. FGF-2-treated CLECs presented diffuse α/9d-reactivity ( Figure 3F), while PLECs had a more defined reactivity for α/9d, highlighting actin filaments in the elongating, differentiating fibre cells ( Figure 3L). LECs treated with only TGF-β2 displayed clear evidence of an EMT response, with strong reactivity for α-SMA, with no β-crystallin observed throughout the explant ( Figure 3D,J).  TGF-β2-treated CLECs had a highly specific localisation of α/9d to actin stress fibres ( Figure 3G), which were also very prominent in PLECs ( Figure 3M). Unlike cells treated with only FGF-2 or only TGF-β2, that had a relatively uniform label for the different markers across the entire explant, in the FGF-2/TGF-β2 cotreated explants, we observed distinct spatial differences in the labelling of the markers, consistent with our earlier morphological observations. The CLECs in the TGF-β2/FGF-2-treated explants predominantly labelled for α-SMA with little to no β-crystallin reactivity at day 3 ( Figure 3E), similar to the explants treated with only TGF-β2 ( Figure 3D,J). In contrast, the PLECs in these same cotreated explants displayed the inverse label, with strong reactivity primarily for β-crystallin in elongated cells, with few neighboring smaller cells immunolabelling for α-SMA ( Figure 3K). This differential β-crystallin and α-SMA reactivity in the cotreated explants was sustained up to 5 days of culture (Supplementary Figure S5). Stronger labelling for α/9d was also observed throughout the cotreated explants ( Figure 3H,N), highlighting the marked elongation of peripheral fibre-like cells ( Figure 3K,N), as well as central myofibroblastic cells (Figure 3E,H).

Western Blotting
Alpha-Smooth Muscle Actin. We quantified protein changes in the treated explants using Western blotting. CLECs had a significant increase in α-SMA when treated with TGF-β2, compared to the relatively lower levels in the control (NT) and FGF-2-treated explants (p < 0.0001) ( Figure 4A,C). FGF-2 treatment did not impact α-SMA levels in the CLECs compared to the control cells (p = 0.3429). In the FGF-2/TGF-β2 cotreated explants, there was a significant reduction in α-SMA levels in the CLECs relative to the TGF-β2 alone CLECs (p < 0.0001). In fact, these CLECs in the cotreated explants displayed no significant difference in levels of α-SMA compared to the CLECs of the control (p > 0.9999) or FGF-2 alone (p = 0.3249) explants. In the PLECs of the FGF-2/TGF-β2 cotreated explants, consistent with the reduced EMT response, there were reduced α-SMA levels when compared to the CLECs, comparable to the lower α-SMA levels seen in the PLECs of the control (p = 0.7371), FGF-2 (p > 0.9999)-, and TGF-β2-treated explants (p = 0.0053) ( Figure 4B,D). The PLECs in the explants treated with FGF-2 alone did not have increased α-SMA levels when compared to the control cells (p = 7366); however, the PLECs in the explants treated with TGF-β2 alone had significantly increased α-SMA levels, compared to the control (p = 0.0203) and the FGF-2-treated (p = 0.0053) explants.
β-crystallin. When compared to the control cells, there was no significant difference in the levels of β-crystallin in the CLECs of the explants treated with FGF-2 (p = 0.8742) ( Figure 4A,C). Treatment with TGF-β2 did not significantly increase levels of β-crystallin in the CLECs compared to the control (p = 0.5844), FGF-2 (p = 0.2260) or the cotreated explants (p = 0.1459). We did observe a significant decrease in β-crystallin in the CLECs of the cotreated explants, relative to the control (p = 0.0160) and FGF-2 (p = 0.0044)-treated explants ( Figure 4A,C). Treatment of the explants with FGF-2 significantly increased βcrystallin levels in the PLECs when compared to the PLECs of the control (p = 0.0374) and the TGF-β2-treated explants (p = 0.0003) ( Figure 4B,D). The PLECs in the explants treated with TGF-β2 alone had reduced β-crystallin levels when compared to the control PLECs (p = 0.0222). The PLECs of the explants cotreated with FGF-2/TGF-β2 had slightly elevated β-crystallin levels in comparison to the PLECs of the control (p = 0.6396) or the TGF-β2-treated (p = 0.0056) explants ( Figure 4B,D).

Discussion
The present study has demonstrated the impact of FGF-2 on the behaviour of lens epithelial cells induced to undergo EMT in response to TGF-β. A lens-fibre-differentiating dose of FGF-2 was able to block TGF-β2-induced lens EMT in only the peripheral LECs in explants (equivalent to the germinative region of the intact lens) and not in the central (more anterior) lens epithelia. As seen in previous wholemount rat lens epithelial cell explant models, we have demonstrated that CLECs and PLECs exposed to TGF-β2 alone undergo an EMT response, with no evidence of lens fibre differentiation [24,41,51]; however, in combination with FGF-2, FGF-2 potentiates this TGF-β2-induced activity, with elevation of canonical Smad2/3 signalling activity, as well as EMT-associated markers, more so in the CLECs.

Discussion
The present study has demonstrated the impact of FGF-2 on the behaviour of lens epithelial cells induced to undergo EMT in response to TGF-β. A lens-fibre-differentiating dose of FGF-2 was able to block TGF-β2-induced lens EMT in only the peripheral LECs in explants (equivalent to the germinative region of the intact lens) and not in the central (more anterior) lens epithelia. As seen in previous wholemount rat lens epithelial cell explant models, we have demonstrated that CLECs and PLECs exposed to TGF-β2 alone undergo an EMT response, with no evidence of lens fibre differentiation [24,41,51]; however, in combination with FGF-2, FGF-2 potentiates this TGF-β2-induced activity, with elevation of canonical Smad2/3 signalling activity, as well as EMT-associated markers, more so in the CLECs.
For our lens epithelial explant model, we used relatively low doses of TGF-β2 (50 and 200 pg/mL) to induce an EMT response across a short culture period [48,52]. This dose is physiologically representative of concentrations of TGF-β2 in its mature (approx. 100 pg/mL) and total (>3000 pg/mL) forms observed in situ [53]. In addition, it is comparable to active forms of TGF-β2 (approx. 100-400 pg/mL) found in cataractous patients [53][54][55][56][57]. Our use of a lower TGF-β2 dose contrasts to other studies that have used much higher doses (0.5-1.5 ng/mL) to elicit an EMT response in rodent lens cells [13,14,16], which could potentially lead to off-target growth factor signalling activity. Exogenous addition of FGF-2 at a high dose encourages all lens epithelial cells (both CLECs and PLECs) to undergo a change in cell morphology typical of fibre differentiation [3,4,58,59]. In explants cotreated with TGF-β2 and FGF-2, FGF-2 appeared to protect PLECs from TGF-β-induced EMT by promoting a fibre differentiation response. We showed that the PLECs in these FGF-2/TGF-β2 cotreated explants had prominent elongated fibres, reminiscent of many earlier studies from our laboratory [59]. An elevated dose of TGF-β2, was able to prevent any fibre differentiation in the PLECs, leading to an enhanced EMT response in both central and peripheral cells. In addition, we demonstrated that the PLECs in the cotreated explants did not exhibit contractile properties as evidenced by the lack of capsular wrinkling in this region, unlike the region of the CLECs undergoing EMT. The inhibition of lens epithelial cell contraction by FGF despite the presence of TGF-β has been shown in other fibrotic models to be dose dependent, such as in bovine LECs cultured in collagen I gel [60] and valvular interstitial cells (VICs) modelling valvular fibrosis [61], which is also correlated with reduced α-SMA expression.
We not only report how TGF-β can impact FGF-induced lens cell responsiveness but how FGF in turn influences TGF-β-induced responses in LECs, the main focus of our study. When we examined for changes in cytoskeletal and stress-fibre associated proteins, the CLECs in TGF-β/FGF-cotreated lens explants exhibited predominant α-SMA localisation and little to no β-crystallin, suggesting that these cells cannot resist the EMT process, despite the presence of a high differentiating dose of FGF-2. The co-influence of FGF-2 and TGF-β on fibre differentiation, epithelial, and EMT-associated marker expression has previously been reported in other models, including human lung epithelial cells and rat alveolar epithelial-like cells [62], as well as E10 chick lens epithelial cells [13], and the lenses of postnatal mice [10]. Despite the CLECs in the TGF-β/FGF-cotreated explants undergoing a prominent EMT response, we noted reduced α-SMA and α/9d levels, compared to the TGF-β-alone-treated explants, suggesting that FGF-2 may be potentially compromising Tpm activity (a recruiter for actin assembly) and attenuating α-SMA stress fibre association. It has been previously shown that FGF may influence Tpm activity and expression, as well as cell biomechanics, in the presence of TGF-β [14]. For example, when murine LECs (MLECs) are cotreated with FGF-2/TGF-β2, the loss of Tpm1 corresponded with decreased α-SMA reactivity [14]. This same study also confirmed FGF-2 modulation of Tpm in HLECs, when cotreated with TGF-β2, with a significant reduction in both Tpm1 and Tpm2 levels [14]. We localized Tpm (α/9d) in LECs undergoing different phenotypic changes in fibre differentiation, but more compellingly in cells undergoing EMT, where it was associated with the α-SMA-reactive stress fibres of myofibroblasts. This may be attributed to the fact that the α/9d antibody we used specifically targets several isoform splice variant products of the αTm gene (TPM1), including Tpm1.4, Tpm1.6-1.9, and Tpm2.1, with some cross-reactivity also for Tpm3.1 [63]. Tpm1.6, Tpm2.1, and Tpm3.1 have all previously been characterized as being stress-fibre associated and are suggested to play a role in TGF-β induced EMT [64][65][66]. FGF has shown a role in propagating stressinduced EMT in conjunction with TGF-β in other pathologies, such as wound healing in mice skin keratinocytes [45] and in the tumor stromal cell microenvironment of prostate fibroblasts [47]. Consistent with our findings, Koike et al. (2020) [45] found that FGF-2 could not solely induce EMT in mice keratinocytes; however, in keratinocytes cotreated with FGF-2 and TGF-β1, there was a significant upregulation of cell migratory/motility and EMT-associated genes (e.g., VIM and SNAI2), similar to keratinocytes with only TGF-β1 stimulation. In a non-transformed mouse mammary gland epithelial cell line (NMuMG), TGF-β modulated FGF receptor activation, increased FGF-2 cell sensitivity, and promoted an EMT response through activation of ERK1/2 signalling [49], highlighting the synergistic signalling role of these two growth factors.
To determine how FGF-2 was modulating and antagonizing TGF-β2-induced EMT in PLECs of cotreated LEC explants, we explored changes in their signalling activity, namely changes to Smad2/3 and ERK1/2. In cotreated lens epithelial explants, we saw stronger signalling for the respective pathways in different regions; CLECs undergoing EMT had more pronounced p-Smad2/3 activity, while PLECs undergoing fiber differentiation had more pronounced p-ERK1/2-signalling. FGF is a well-known regulator of ERK1/2 within the lens, with its marked phosphorylation evident in lens cells within minutes post treatment [3,4,67,68]. While ERK1/2 has been shown to be required for lens epithelial cell proliferation, it is also very important for lens fiber differentiation [4,[67][68][69][70]. This differs from TGF-β2-induced EMT, where we found that while ERK is also involved in this EMT process, blocking ERK1/2 does not completely block TGF-β2-mediated EMT progression in lens epithelia [39,48,52]. In fact, canonical Smad2/3-signalling is most evident in EMT, as shown here in our cotreated CLECs, and in many earlier studies examining TGF-β2induced lens EMT [11,13,38,52,71,72].
While we and others have shown FGF-2 is not able to promote Smad2/3-signalling in LECs [10], FGF-2 was shown to impede nuclear localisation of Smad2/3 in PLECs in explants cotreated with TGF-β2; however, in CLECs of these same explants, FGF-2 appeared to have less of an impact on TGF-β2-induced Smad2/3-activity. How FGF-2 directly blocks Smad2/3 activity in PLECs is not clear but given the strong ERK1/2 activation in these cells, this may favour lens fibre differentiation and cell survival, as we see here and has been shown by others [2,3,67,73]. Conversely, FGF-mediated ERK1/2 signalling can correlate with the upregulation of TGF-β activity, as seen in other fibrosis models [14,49,[74][75][76], as well as the current study where TGF-β-induced CLECs are associated with elevated ERK1/2-signalling. Similar to the current study, in valvular interstitial cells (VICs) modelling valvular fibrosis, it was shown that inhibition of this fibrosis was dependent on FGF-2-mediated MAPK signalling when cotreated with TGF-β1 [61]. This study demonstrated that FGF (10 ng/mL) prevented Smad3 nuclear localisation in VICs cotreated with TGF-β1 (5 ng/mL), and at higher doses (100 ng/mL), it was able to perturb TGF-β1-mediated α-SMA expression [61], highlighting the ability of FGF to modulate canonical TGF-β signalling activity and downstream gene expression.
Although not completely understood, crosstalk between FGF and TGF-β signalling has proven influential in mediating various fibrotic disorders and carcinoma progression. For example, a study implementing mouse tumor-associated endothelial cells (TECs) demonstrated how FGF can promote a differential cell response by reducing TGF-β-induced contractile and myofibroblastic properties, while concurrently promoting cell proliferation and motility [75]. A similar finding was observed in primary human dermal fibroblasts (HDFs), whereby FGF-2 with TGF-β1 cotreatment, both positively and negatively regulated fibroblast transition into cancer-associated fibroblasts (CAFs) [77]. This same study also showed how this FGF-2/TGF-β1 treatment of HDFs can downregulate common CAFactivated and EMT-associated markers (e.g., ACTA2, ITGA11, and COL1A1) as well as upregulating cell motility and morphogenetic genes (e.g., HGF and BMP2) [77].
Research into the mechanisms surrounding differential types of PCO involving lens fibre cell types is ongoing and it is believed to be due to FGF/TGF-β interactions during EMT induction [13,15,16,38,74,78]. As FGF is a major factor influencing normal lens fibre differentiation, it is important to understand what promotes aberrant fibre differentiation during pearl PCO development at the lens equator [13,24,35,79]. In situ, for ASC and for post-operative PCO, more anterior lens epithelial cells are likely exposed to a high insult of TGF-β, and relatively low levels of FGF are normally found in the aqueous humour. At the lens equator, however, epithelial cells in the posterior chamber are regularly exposed to elevated levels of FGF, and regardless of any increased TGF-β levels, the cells here likely undergo aberrant fibre differentiation, leading to pearl PCO. This may result from the heightened sensitivity to FGF of these peripheral LECs, namely due to their elevated levels of high-affinity FGF receptor tyrosine kinase (RTK) receptors, compared to the central lens epithelia [6,58,80,81]. In situ, during lens fibrosis, we do not see EMT resulting in cell death, likely due to survival growth factors present within the ocular media. Given the findings from the current study, we propose that FGF is a putative survival factor in situ, maintaining fiber cells at the lens equator and the myofibroblastic phenotype leading to fibrotic PCO. Further studies investigating differences/changes in levels of FGF and TGF-β receptors, between central and peripheral lens cells in cotreated explants, may be a key factor in determining lens cell fates in situ. We also cannot rule out that changes in the expression of RTK antagonists, such as Sprouty and Spreds [69,70,82], including those more specific for FGF, such as Sef [83], in these active regions of the lens may be protective of peripheral LECs from any aberrant TGF-β insult of which they have previously been reported to block [69].

Conclusions
A fine balance between levels of FGF-2 and TGF-β2 can promote differential responses in lens epithelial cells. More specifically, this responsiveness is spatially regulated, with the anterior central lens epithelia more sensitive to EMT induction, whilst peripheral cells primarily undergo fibre differentiation in the presence of high levels of FGF, avoiding apoptotic cell death associated with EMT. This induced heterogeneous population of cells in lens epithelial explants may provide an alternative model better suited to the study of the cellular processes at play in situ, leading to the formation of ASC, and more importantly both fibrotic and pearl forms of PCO.