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E. Felinski, D. A. Antonetti; A Novel Tight Junction Promoter Element Controls Steroid Induction of the Blood-Retinal Barrier. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3989. doi: https://doi.org/.
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Tight junction protein complexes between endothelial cells in retinal blood vessels contribute to 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 (GC) induce barrier properties of the retinal microvasculature through a mechanism that involves increased tight junction protein expression. In this study we investigated the dose response and time course of GC induction of the endothelial barrier in vitro and identified a novel GC-responsive cis-element in the occludin gene promoter.
Primary bovine retinal endothelial cells (BREC) in culture were treated with GC and assayed for changes in barrier properties and for changes in occludin and claudin-5 protein content. Occludin promoter-luciferase and claudin-5-luciferase plasmid constructs were transfected into BREC and luciferase activity was measured with and without GC treatment. Deletion and point-mutational analysis was used to identify the minimally responsive fragment of the occludin promoter and electrophoretic mobility shift assays (EMSA) assays were used to identify DNA binding proteins.
Endothelial barrier induction by dexamethasone was maximal with a dose of 50 ng/ml for 48 hrs. Increased occludin and claudin-5 protein expression, as well as increased promoter activity, was observed at 24 hrs. Mapping of the occludin promoter element responsible for GC induction revealed a 40 bp occludin enhancer element (OEE), which was sufficient to confer GC-stimulated transcription in BREC. This cis-element does not directly bind the glucocorticoid receptor, rather in vitro binding studies demonstrate specificity for a novel trans-acting factor from BREC nuclear extracts.
The results demonstrate that GC treatment of retinal endothelial cells increases barrier properties in vitro and directly increases tight junction protein expression through gene activation. GC induction of the occludin gene promoter functions through a novel enhancer element, the OEE, conserved in claudin-5, that may co-ordinate gene expression of the BRB.
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