Abstract
Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration.
Highlights
Retinal function is dependent on the retinal pigment epithelium (RPE), which is a monolayer of tightly connected pigmented cells underlying the photoreceptor cell layer
We demonstrate that alteration of the levels of the tight junction (TJ) components zonula occludens (ZO)-1 and ZONAB leads to changes in RPE cell proliferation, differentiation and function
Downregulation of ZO-1 or the overexpression of ZONAB led to the induction of cell proliferation and altered morphology correlating with expression of five epithelial-mesenchymal transition (EMT) markers: glial fibrillary acidic protein (GFAP), vimentin, N-cad, Snail1 and cyclin D1 (cD1)
Summary
Retinal function is dependent on the retinal pigment epithelium (RPE), which is a monolayer of tightly connected pigmented cells underlying the photoreceptor cell layer. Tight junctions (TJs) are a type of cell-cell adhesion that have a fundamental role for the BRB function because they regulate paracellular diffusion across epithelia [4]. They separate apical and lateral membrane components, and take part in signalling pathways involved in epithelial proliferation, gene expression and differentiation [5,6]. ZONAB expression decreased and inversely correlated with increasing apical differentiation, reflected by maturation of the brush border and extension of the primary cilium [13]. The effect of ZONAB overexpression on differentiation has not been shown yet in vivo
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