Citation: Flokis, M.; Lovicu, F.J. FGF-2 Differentially Regulates Lens Epithelial Cell Behaviour during TGF-β-Induced EMT. Cells 2023, 12, 827. https://doi.org/10.3390/ cells12060827 Academic Editor: Alexander V. Ljubimov Received: 14 February 2023 Revised: 2 March 2023 Accepted: 6 March 2023 Published: 7 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article FGF-2 Differentially Regulates Lens Epithelial Cell Behaviour during TGF-β-Induced EMT Mary Flokis 1 and Frank J. Lovicu 1,2, * 1 Molecular and Cellular Biomedicine, School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW 2006, Australia 2 Save Sight Institute, The University of Sydney, Sydney, NSW 2006, Australia * Correspondence: frank.lovicu@sydney.edu.au Abstract: 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 mini- mal 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 regu- lating 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. Keywords: transforming growth factor-beta (TGF-β); fibroblast growth factor (FGF); epithelial-mesenchymal transition (EMT); fibrosis; cataract; lens 1. Introduction The ocular lens is a transparent, avascular tissue responsible for transmitting light onto the retina. It contains two cell types: cuboidal epithelial cells and adjacent elongate fibre cells, both comprised of specialized molecular (e.g., crystallins) and cytoskeletal (e.g., intermediate filaments) properties to facilitate vision [1]. Ocular growth factors, such as fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-β), are key regulators of different cellular processes in the lens, including epithelial cell proliferation [2–4], fibre differentiation [1,5–10], and epithelial–mesenchymal transition (EMT) that lead to lens pathology [11–17]. In situ, FGF is thought to be required for regulat- ing normal lens cell processes in a spatially dependent manner, as previously reviewed [1]. Cells 2023, 12, 827. https://doi.org/10.3390/cells12060827 https://www.mdpi.com/journal/cells