ITF2357, a histone deacetylase inhibitor protects barrier function in polarized ARPE-19 cells

Abstract

Purpose: To study the role of histone deacetylase inhibitor (HDACi), ITF2357 on the integrity of the barrier function in the TNFα-treated human retinal pigment epithelial (ARPE-19) cells.<br />

Methods: Transepithelial electrical resistance (TEER) was measured in the polarized ARPE-19 cells grown on a transwell dishes and treated with TNFα (10 ng/mL) in the presence of different doses of ITF2357 (0.25 μM, 1.0 μM and 2.5 μM). ITF2357 was tested in ARPE-19 cells for safety, efficacy and toxicity using xCELLigence real time monitoring, Prestoblue assay, MTT assay and TUNEL assay. Permeability assay was performed using sodium fluorescein dye. Real-time PCR, immunostaining and western blot was performed to study the tight junction (ZO-1, ZO-2, ZO-3, occluding, Claudin 1, Claudin-3, Claudin-5, Cloudin-10) and adhesion proteins (α-catenin, β-catenin, γ-catenin, E-cadherin, N-cadherin, Nestin and Afadin). Scanning electron microscopy (SEM) and Transmission electron microscopy was used to determine the ultrastructure of the tight-junctions in the cells. NFkB signaling was studied using acetyl NFkB antibody and transcription assay using ELISA kit. <br /> <br />

Results: TNFα treatment decreased the TEER in the ARPE-19 cells, which can be rescued in the pre-treated ITF2357 cells. ITF2357 hyperacetylates and reduced the permeability of the ARPE-19 cells. ITF2357 activates tight and adheren junction proteins in the presence of TNFα-treated ARPE-19 cells as observed by real-time PCR and western blotting. ITF2357 attenuates phosphorylation of RelA proteins and increases the transcription of negative regulator-IkBα in the ARPE-19 cells.

Conclusions: <br /> <br /> ITF2357 significantly increased majority of tight and adherens junction proteins and regulates NFkB signaling to protect outer blood-retina barrier integrity in TNFα-treated polarized ARPE-19 cells.