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Shannon Das, Frank J Lovicu; TGFß-induced EMT leading to cataractogenesis involves Nox4 activity. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1120. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Transforming Growth Factor-β (TGFβ) can induce an epithelial to mesenchymal transition (EMT) in lens that results in fibrotic cataract. We and others have previously shown that the reactive oxygen species (ROS) producing enzyme, NADPH oxidase 4 (Nox4), is upregulated in response to TGFβ-signalling in lens, and that pharmacological inhibition of Nox4 can abrogate aspects of this EMT. To better elucidate the role(s) of Nox4 in lens EMT in situ, the present study characterised TGFβ-induced cataract formation in the absence of Nox4.
We crossed mice overexpressing TGFβ in the lens, that develop anterior subcapsular cataract, with Nox4-deficient mice. The eyes of resultant progeny were grossly examined, collected and processed for histological evaluation. Eye sections were stained with Periodic-acid Schiff, and immunofluorescence was used to assess changes in epithelial and EMT/cataract markers. Western blotting for pERK1/2, pSmad2/3 and Nox4, and qPCR for Nox subunits were performed on lens epithelial explants from different mice (wild-type-WT, Nox4-deficient and/or TGFβ-overexpressing). Additionally, lens epithelial explants from WT or Nox4-deficient mice were treated with exogenous TGFβ2 (200pg/ml) and stained for reactive oxygen species using DHE and Mitosox.
Mice overexpressing TGFβ deficient for Nox4, did not present anterior subcapsular cataracts by postnatal day 30, with the lens remaining transparent. Despite this transparency, histology and immunolabelling of lenses revealed the presence of anterior subcapsular plaques devoid of many EMT markers. Labelling of Nox4-deficient lens epithelial explants and mouse sections revealed elevated pSmad2/3 and pERK1/2 signalling. qPCR analysis revealed compensatory upregulation of Nox2 in mice deficient for Nox4. Moreover, TGFβ-treatment induced elevated but delayed ROS labelling in Nox4-deficient explants.
These results indicate that in mice at least, Nox4-induced ROS plays a role in the development of TGFß-induced EMT leading to cataract, potentially by modulating pSmad2/3 and pERK1/2 signalling. In the absence of Nox4, there are other compensatory sources of ROS, such as Nox2 and mitochondrial ROS. Taken together, these findings provide a better understanding of TGFß-induced EMT, as well as a platform allowing us to delineate putative Nox4 interactions with Smad2/3 and/or ERK1/2, as well as other signalling intermediates.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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