ITF2357 inhibits NF-kB signaling pathway to regulate barrier integrity in retinal pigment epithelial cells

Abstract

Purpose : To study the mechanism of action of a histone deacetylase inhibitor, ITF2357 in the regulation of barrier function in TNFα-treated retinal pigment epithelial cells

Methods : Human retinal pigment epithelial cells (ARPE-19) were polarized on transwell dishes and treated with TNFα (10 ng/mL) in the presence of ITF2357 (2.5 μM) to measure barrier integrity using transepithelial electrical resistance (TEER) and epithelial cell permeability using FITC-dextran. Kinetics of NFkB signaling was evaluated in the cytosolic and nuclear cellular fractions using western blots and NFkB activity assay using ELISA kit. Immunostaining was performed to study nuclear shuttling of p65. Real-time PCR, immunostaining and western blots were performed to study the tight junction (ZO-1, ZO-2, ZO-3, occludin, Claudin 1, Claudin-3, Claudin-5, Claudin-10) and adhesion junction proteins (α-catenin, β-catenin, γ-catenin, E-cadherin, N-cadherin, Nestin and Afadin) . This was supported by ultrastructure studies using scanning electron microscopy (SEM) and transmission electron microscopy .

Results : ITF2357 restores barrier function and inhibits TNFα-induced decrease in TEER and increase in permeablility observed in polarized retinal pigment epithelial cells. ITF2357 also reinstate tight and adherens junction proteins at the transcript and protein levels in TNFα-treated cells. ITF2357 attenuates phosphorylation of p65 and its translocation into the nucleus. We found that ITF2357 regulates phosphorylation of IKK and transcriptionally modulates negative regulator-IkBα turnover in the ARPE-19 cells. In addition, ITF2357 also restores tight junctions as observed by TEM.

Conclusions : We conclude that ITF2357 protects barrier integrity via regulation of NF-kB signaling components in the retinal pigment epithelial cells and might serve as a potential drug for the treatment of retinal diseases involving outer blood-retina barrier function.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.