Nox4-mediated ROS production is involved, but not essential for TGFβ-induced lens EMT leading to cataract

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Highlights

  • Nox4 is not essential but involved in TGFβ-induced lens EMT in situ.

  • Nox4 deficiency delays the onset of TGFβ-induced cataract in situ.

  • In situ, Nox2 upregulation may compensate for a loss of Nox4 expression.

  • ERK1/2-signaling is required for TGFβ/Smad2/3-signaling.

Abstract

The reactive oxygen species (ROS) producing enzyme, NADPH oxidase 4 (Nox4), is upregulated in response to TGFβ in lens epithelial cells in vitro, and its selective inhibition was shown to block aspects of TGFβ-induced epithelial-mesenchymal transition (EMT). In the present in situ study we validate the role(s) of Nox4 in TGFβ-induced lens EMT leading to anterior subcapsular cataract (ASC) formation. Mice overexpressing TGFβ in the lens, that develop ASC, were crossed to Nox4-deficient mice. When comparing mice overexpressing TGFβ in lens, to mice that were also deficient for Nox4, we see the delayed onset of cataract, along with a delay in EMT protein markers normally associated with TGFβ-induced fibrotic cataracts. In the absence of Nox4, we also see elevated levels of ERK1/2 activity that was shown to be required for TGFβ/Smad2/3-signaling. qRT-PCR revealed upregulation of Nox2 and its regulatory subunit in TGFβ-overexpressing lens epithelial cells devoid of Nox4. Taken together, these findings provide an improved platform to delineate putative Nox4 (and ROS) interactions with Smad2/3 and/or ERK1/2, in particular in the development of fibrotic diseases, such as specific forms of cataract.

Introduction

Cataract, the opacification of the eye lens, is one of the major causes of blindness worldwide (Pascolini and Mariotti, 2012). Due to its progressive nature and the absence of effective non-invasive treatment strategies, surgery remains the only option for patients with hampered visual acuity (Linebarger et al., 1999). Cataract surgery, while removes much of the lens cellular content, results in residual lens epithelial cells (LECs) remaining attached to the lens capsule. As part of a wound healing response, these cells proliferate and migrate to progressively populate areas of the denuded lens capsule, as they undergo an epithelial to mesenchymal transition (EMT) into contractile myofibroblastic cells that modulate and deposit aberrant extracellular matrix (ECM) (Liu et al., 1994; Wojciechowski et al., 2017). This aggregation of mesenchymal growths across the posterior aspect of the lens capsule contributes to a secondary cataract, more commonly known as posterior capsular opacification (PCO) (Eldred et al., 2011).

A concerted effort has been made in understanding the molecular underpinnings of EMT, in the hope to identify potential interventional targets. Studies have identified Transforming Growth Factor-beta (TGFβ) as an inducer of EMT and fibrotic forms of cataract. Both in vitro (Das et al., 2016; Liu et al., 1994; Shu et al., 2018; Wojciechowski et al., 2017) and in vivo (Saika et al., 2004; Srinivasan et al., 1998) studies have shown that TGFβ can induce a fibrotic response in LECs that is marked by their elongation, their acquisition of contractile activity, as well as their modification of the underlying lens capsule. More recently, research has focused on identifying a role for oxidative stress in the onset of TGFβ-induced EMT leading to fibrotic cataract. Chamberlain and colleagues were the first to identify a role for reactive oxygen species (ROS) in EMT of the lens (Chamberlain et al., 2009). Using both whole lenses and lens epithelial explants, concomitant treatment of TGFβ with antioxidants, either glutathione (GSH) or catalase, blocked the fibrogenic effects of TGFβ (Chamberlain et al., 2009), suggestive of an important role of ROS in the progression of EMT in these cells. This is in congruence to findings from mouse LECs that were depleted of GSH synthesis pathways that showed increased pro-EMT markers (Wei et al., 2017). These findings provide important evidence that alludes to a distinct function of TGFβ in the production of ROS in LECs.

One of the potential sources of ROS production is via the NADPH Oxidase (Nox) family of proteins (Bedard and Krause, 2007). One specific Nox member, Nox 4 has been reported to be markedly increased in LECs treated with TGFβ (Das et al., 2016). Our laboratory has previously established that pharmacological inhibition of this Nox member delayed the progression of EMT and abrogated the development of myofibroblasts in vitro (Das et al., 2016). Nox4 protein levels were found to increase and be dependent on TGFβ at early stages of lens EMT; however, more importantly, Nox4 levels continued to be elevated later in the culture period (up to 48 h) when the morphological changes of EMT are more noticeable in this lens explant model. Nox4 was found to only partially block the morphological progression of lens EMT, and this was suggestive of only part suppression of Nox4 activity with the selective inhibitors, or that there were other yet to be identified sources of ROS at play. Given that the currently available pharmacological inhibitors of Nox4 often lack isoform specificity, and this remains one of the drawbacks for both experimental and clinical use (Laleu et al., 2010), here we use a gene targeted approach to examine TGFβ-induced lens EMT in situ in the absence of Nox4.

Murine Nox4-deficient models have been used extensively to explore potential roles for Nox4 in fibrosis, nephropathy and cardiac disease (Babelova et al., 2012; Braunersreuther et al., 2013; Sirokmany et al., 2016), and have been associated with significant rescuing effects in numerous disease models (Schroder et al., 2009; Sirokmany et al., 2016). In some other models; however, Nox4 deletion has been reported to have negligible impact on disease progression (Babelova et al., 2012). This latter point reiterates the pleotropic nature of ROS targets and raises the question of exactly what role Nox4 plays in the lens, and whether other sources of ROS are involved in TGFβ-induced lens EMT. To that avail, the present study aims to further characterise the role of Nox4 in lens EMT by utilising a transgenic mouse line overexpressing TGFβ specifically in the lens that presents anterior subcapsular cataract (ASC) (Srinivasan et al., 1998), on a Nox4-deficient background. Here we show the importance of Nox4 in maintenance of the lens epithelial phenotype, and establish a role for Nox4 in the onset of TGFβ-induced EMT leading to fibrotic cataract. Moreover, alternate sources of ROS in the absence of Nox4 during this TGFβ-induced EMT are explored as putative compensatory mechanisms.

Section snippets

Materials and methods

All handling and management of animals were conducted in accordance with the National Health Medical Research Council (Australia) guidelines, and the Association for Research in Vision and Ophthalmology Handbook for the Use of Animals in Biomedical Research, USA. The Animal Ethics Review Committee of The University of Sydney, NSW, Australia, approved all procedures.

Nox4 is involved in TGFβ-induced cataractogenesis

To determine a role for Nox4 in the development of TGFβ-induced cataract in situ, we compared lenses of TGFβGOF transgenic mice, with or without a deficiency for Nox4. Isolated lenses from enucleated eyes of P30 mice were qualitatively examined for the presence/absence of opacities. Compared to the transparent lenses of WT mice (Fig. 1A), TGFβGOF lenses were opaque, presenting a large centrally positioned anterior subcapsular plaque (Fig. 1B). Nox4-deficient lenses were transparent, similar to

Discussion

In the present study we have shown that a deficiency of Nox4 in LECs is not sufficient to prevent TGFβ-induced cataractogenesis. At P30, both TGFβGOF and TGFβGOF/NKO lenses exhibited histological anterior subcapsular plaques that were characterised by multilayering of the lens cells with elongate cell nuclei. Interestingly, only the TGFβGOF lenses appeared opaque upon gross examination, attributed to the fact that the plaques of the TGFβGOF mice lens were more heterogenous; containing

Acknowledgements

This project was supported by the NWG Macintosh Memorial Fund, Discipline of Anatomy & Histology, The University of Sydney (SD and FJL). SD was supported through an Australian Postgraduate Award scholarship.

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