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E. Felinski, J. Keil, D. Antonetti; Glucocorticoids Induce p54/NonO and Proline/Glutamine Rich Splicing Factor (PSF) Binding to a Novel Cis-Element Coordinating Junctional Protein Expression. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5097.
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The endothelial tight junction complex contributes to formation of the blood-retinal barrier (BRB) and loss of this barrier contributes to a number of retinal diseases. Occludin and claudin-5 are transmembrane tight junction proteins essential for the establishment and maintenance of the vascular barrier and previous studies have demonstrated that glucocorticoids (GC) increase transcription of these genes through activation of an occludin enhancer element (OEE). The purpose of these studies was to determine the mechanism of transcriptional control of occludin and claudin-5 by identifying GC-induced OEE-binding factors in retinal endothelial cells.
Primary human retinal endothelial cells (HREC) were treated with GC and nuclear extract was used to purify OEE-binding factors by DNA-affinity purification (DAP). Transcription factors were identified by tandem MALDI-TOF MS/MS mass spectrometry. GC-induced binding was assessed by DAP followed by western blot. Chromatin immunoprecipitation (ChIP) was used to analyze DNA/protein interactions in cell culture and in vivo.
MS/MS analysis of GC-treated HREC nuclear extracts identified the transcription factors p54/NonO and PSF, known to act as a heterodimeric coactivator, bound to the OEE. DAP demonstrated increased binding of p54/NonO and PSF from GC-treated HREC nuclear extracts compared to controls. ChIP assay confirmed p54/NonO and PSF binding in cells to the occludin and claudin-5 promoters as well as the cadherin-9 promoter, which also possesses an OEE.
p54/NonO and PSF bind to the OEE of the occludin and claudin-5 gene promoters in a GC-dependent manner. The heterodimer also associates with a putative OEE in the steroid-responsive cadherin-9 promoter. Identification of the factors that control transcription from the OEE will elucidate how GC stimulate the blood-retinal barrier, potentially leading to novel therapeutic targets that induce barrier properties.
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