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Jason M. Keil, Edward A. Felinski, David A. Antonetti; Glucocorticoid-Induction of The Blood-Retinal Barrier Requires Transcription Factor p54 Non-octet. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4846.
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Loss of the blood-retinal barrier (BRB) in diabetic retinopathy may be reversed with glucocorticoids although refractiveness and complications make this therapeutic option unfavorable. Understanding the mechanism by which glucocorticoids induce BRB properties may provide novel therapies without these complications. Previous work identified the occludin enhancer element (OEE) as a glucocorticoid-responsive cis-element in the promoters of the tight junction genes occludin and claudin-5. Here, we identify the OEE binding factors and determine their contribution to glucocorticoid-induction of tight junction gene expression and endothelial barrier properties.
OEE-binding factors were isolated from human retinal endothelial cells (HREC) using DNA affinity purification followed by MALDI-TOF MS/MS. Chromatin immunoprecipitation (ChIP) assays demonstrated trans-acting factor binding to DNA in cells and tissue. Transfection of siRNA was used to evaluate the role of trans-acting factors in the transcription of tight junction genes occludin and claudin-5 in response to glucocorticoid stimulation in HREC and bovine retinal endothelial cells (BREC). Protein levels were quantified by Western blot. Paracellular permeability was determined by quantifying the flux of 70 kDa RITC-Dextran through a cell monolayer on Transwell filters. Transendothelial electrical resistance (TER) was measured in real time utilizing the ECIS system.
MS/MS analysis of HREC nuclear extracts identified the heterodimer of transcription factors p54/NonO and pro/gln rich splicing factor (PSF) as OEE-binding factors, which was confirmed by ChIP assay. Specific siRNA knockdown of p54 demonstrated that this factor is necessary for glucocorticoid induction of occludin and claudin-5 gene expression. Further, p54 knockdown ablated the glucocorticoid-induced decrease in dextran flux and increase in TER. Finally, an in vivo ChIP assay demonstrated that these factors are bound to the occludin and claudin-5 gene promoters in the retina.
The p54/NonO and PSF heterodimer is essential for glucocorticoid-mediated expression of occludin and claudin-5 and barrier induction. These same factors may contribute to normal blood-retinal barrier induction in vivo.
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