β-Catenin/Smad3 Interaction Regulates Transforming Growth Factor-β-Induced Epithelial to Mesenchymal Transition in the Lens.

Aftab Taiyab,Judith A West-Mays,Julie Holms

doi:10.3390/ijms20092078

Aftab Taiyab, Judith A West-Mays + Show 1 more

Open Access

https://doi.org/10.3390/ijms20092078

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Abstract

Cataracts are the leading cause of blindness worldwide. Although surgery is a successful method to restore vision loss due to cataracts, post-surgical complications can occur, such as secondary cataracts, also known as posterior capsular opacification (PCO). PCO arises when lens epithelial cells (LEC) are left behind in the capsular bag following surgery and are induced to undergo epithelial to mesenchymal transition (EMT). Following EMT, LEC morphology and phenotype are altered leading to a loss of transparency and vision. Transforming growth factor (TGF)-β-induced signaling through both canonical, TGF-β/Smad, and non-canonical, β-catenin/Wnt and Rho/ROCK/MRTF-A, pathways have been shown to be involved in lens EMT, and thus PCO. However, the interactions between these signaling pathways in the lens have not been thoroughly explored. In the current study we use rat LEC explants as an ex vivo model, to examine the interplay between three TGF-β-mediated pathways using α-smooth muscle actin (α-SMA) as a molecular marker for EMT. We show that Smad3 inhibition via SIS3 prevents nuclear translocation of β-catenin and MRTF-A, and α-SMA expression, suggesting a key role of Smad3 in regulation of MRTF-A and β-catenin nuclear transport in LECs. Further, we demonstrate that inhibition of β-catenin/CBP interaction by ICG-001 decreased the amount of phosphorylated Smad3 upon TGF-β stimulation in addition to significantly decreasing the expression levels of TGF-β receptors, TBRII and TBRI. Overall, our findings demonstrate interdependence between the canonical and non-canonical TGF-β-mediated signaling pathways controlling EMT in the lens.

Highlights

Cataract, a loss in transparency of the ocular lens, continues to be the leading cause of blindness worldwide, affecting nearly 20 million people, with the incidence rising due to a global ageing population [1,2,3]
Fibrotic cataract or fibrosis of the ocular lens is primarily caused by epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) that results in lens opacification and loss of vision
The current study focuses on understanding the interaction of Transforming growth factor (TGF)-β-stimulated Smad3 and β-catenin signaling during EMT of LEC explants

Summary

Introduction

A loss in transparency of the ocular lens, continues to be the leading cause of blindness worldwide, affecting nearly 20 million people, with the incidence rising due to a global ageing population [1,2,3]. Transforming growth factor β (TGF-β), a major cytokine, has been shown to induce EMT-driven fibrosis [5] in many systems such as lungs [10], kidney [11] and heart [12] Both active and latent forms of TGF-β have been detected in the aqueous humor of the anterior chamber of the eye, and have been implicated in EMT-mediated lens fibrotic disorders including ASC and PCO [13,14] in response to surgical or accidental injury [15,16,17,18,19]. We seek to determine whether TGF-β-induced Smad activation is β-catenin dependent and necessary for nuclear translocation of MRTF-A, and if Smad is critical for delocalization of β-catenin during TGF-β-induced EMT in the lens

Smad3 Signaling Inhibition Blocks TGF-B-Induced EMT in Lens Explants

Reagents

Culturing Rat Lens Epithelial Explants

Treatment of Lecs with TGF-B and Inhibitors

Western Blot Analyses

Immunocytochemistry