Glucocorticoid Treatment Regulates Promoter Activity of the Tight Junction Genes Occludin and Claudin–5 in Retinal Vascular Endothelial Cells

Abstract

Abstract: : Purpose: Tight junction protein complexes between endothelial cells in retinal blood vessels provide the blood–retinal barrier. Breakdown of this barrier is problematic in several disease states including diabetic retinopathy. Occludin and members of the claudin family are transmembrane tight junction proteins essential for the establishment and maintenance of the vascular barrier and occludin has been shown to decrease in diabetic retinas. Glucocorticoids induce barrier properties of the retinal microvasculature through a mechanism that involves increased tight junction protein expression. This study set out to determine the pathway by which glucocorticoids induce tight junction protein expression in order to develop new therapies for diabetic retinopathy. Methods: Primary bovine retinal endothelial cells (BREC) in culture were treated with glucocorticoids and assayed for changes in occludin and claudin–5 protein content and for changes in barrier properties. The glucocorticoid receptor antagonist RU486 was used to determine if a transactivation or transrepression mechanism is responsible for the observed effects. The upstream promoter regions of both the occludin and claudin–5 genes were cloned into luciferase reporter plasmids and transfected into BREC. Promoter activity was assessed with and without glucocorticoid treatment. Results: Treatment of BREC with 50 ng/ml dexamethasone for 24 hr increased occludin and claudin–5 protein content and decreased the permeability of a BREC monolayer to 70 kD RITC–dextran. RU486 completely reversed these glucocorticoid effects suggesting a gene transactivation mechanism is responsible. Occludin and claudin–5 luciferase promoter constructs showed a 2–3 fold increase in activity upon transfection into BREC and dexamethasone treatment. Conclusions: The results demonstrate that glucocorticoid treatment of retinal endothelial cells directly increases the expression of the genes for the tight junction proteins occludin and claudin–5 through increased promoter activity. The lack of a canonical GRE in the gene promoters suggests that either a non–canonical cis–element is employed, or induction of a secondary element is necessary.

Keywords: diabetic retinopathy • transcription • vascular cells