Claudins regulate gene and protein expression of the retinal pigment epithelium independent of their association with tight junctions

Abstract

Claudin-19 is the major claudin in the tight junctions of the retinal pigment epithelium (RPE). Claudin-3 is also uniformly expressed albeit in lesser amounts. Besides modulating transepithelial diffusion, claudins modulate gene expression. The absence of claudin-19 and claudin-3 in the RPE cell lines, ARPE-19 and hTERT-RPE-1, provide an opportunity to examine whether exogenous claudins regulate gene expression in the absence of tight junctions. Quantitative RT-PCR was used to compare gene expression in ARPE-19 and hTERT-RPE-1 with that of highly differentiated, human fetal RPE. Claudin-19 and claudin-3 were exogenously expressed using an adenoviral vector. The transepithelial electrical resistance (TER) was measured using Endohm electrodes, and the effects of claudin on the actin cytoskeleton were determined by immunocytochemistry. The effect of claudin on gene expression was examined by quantitative RT-PCR and western blotting. Aside from claudin-19 and claudin-3, ARPE-19 and hTERT-RPE-1 expressed most junction-associated mRNAs in amounts comparable to human fetal RPE, but some RPE signature and maturation genes were under-expressed. Unlike ARPE-19, hTERT-RPE-1 failed to form tight junctions or develop a TER. Claudins exogenously expressed in hTERT-RPE-1 failed to crystalize an apical junctional complex. Actin filaments were not redistributed from stress fibers to cortical bands, and a TER was not established. In hTERT-RPE-1, claudins were found only in internal vesicular-like structures. Nonetheless, claudins increased the expression of the mRNAs for a collection of RPE-enriched proteins. Claudin-19 and claudin-3 had different effects on gene and protein expression indicating activation of overlapping, but distinct, signaling pathways. A major difference was the ability of claudin-19 to affect steady-state levels of ADAM9 and tyrosinase in ARPE-19. In conclusion, claudins can increase the barrier function of a pre-existing apical junctional complex, but on its own it cannot recruit tight junction proteins to form a complex de novo. Many effects of claudin on gene expression did not require an association with the apical junctional complex. Although claudin-19 shared many effects with claudin-3, claudin-19 exerted unique effects on the maturation of RPE.

Keywords: Claudin-19; Claudin-3; Gene expression; Retina; Retinal pigment epithelium; Tight junctions.