title: Glucocorticoid induction of occludin expression and endothelial barrier requires transcription factor p54 NONO.
abstract: OBJECTIVE
  Glucocorticoids (GCs) effectively reduce retinal edema and induce vascular barrier properties but possess unwanted side effects. Understanding GC induction of barrier properties may lead to more effective and specific therapies. Previous work identified the occludin enhancer element (OEE) as a GC-responsive cis-element in the promoters of multiple junctional genes, including occludin, claudin-5, and cadherin-9. Here, we identify two OEE-binding factors and determine their contribution to GC induction of tight junction (TJ) gene expression and endothelial barrier properties.
  
  
  METHODS
  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 determined in situ binding. siRNA was used to evaluate the role of trans-acting factors in transcription of TJ genes in response to GC stimulation. Paracellular permeability was determined by quantifying flux through a cell monolayer, whereas transendothelial electrical resistance (TER) was measured using the ECIS system.
  
  
  RESULTS
  MS/MS analysis of HREC nuclear extracts identified the heterodimer of transcription factors p54/NONO (p54) and polypyrimidine tract-binding protein-associated splicing factor (PSF) as OEE-binding factors, which was confirmed by ChIP assay from GC-treated endothelial cells and rat retina. siRNA knockdown of p54 demonstrated that this factor is necessary for GC induction of occludin and claudin-5 expression. Further, p54 knockdown ablated the pro-barrier effects of GC treatment.
  
  
  CONCLUSIONS
  p54 is essential for GC-mediated expression of occludin, claudin-5, and barrier induction, and the p54/PSF heterodimer may contribute to normal blood-retinal barrier (BRB) induction in vivo. Understanding the mechanism of GC induction of BRB properties may provide novel therapies for macular edema.
authors: Department of Ophthalmology and Visual Sciences, University of Michigan Kellogg Eye Center, Ann Arbor, Michigan, USA.
authors: Jason M
authors: JM
authors: Keil
authors: Xuwen
authors: X
authors: Liu
authors: David A
authors: DA
authors: Antonetti
mesh_terms: D000818:Animals; D001813:Blood-Retinal Barrier / Q000187:drug effects* / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D062507:Claudin-5 / Q000378:metabolism; D004268:DNA-Binding Proteins; D003907:Dexamethasone / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D020868:Gene Silencing / Q000502:physiology; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D008297:Male; D034521:Nuclear Matrix-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D062793:Occludin / Q000096:biosynthesis*; D050810:Octamer Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000072670:PTB-Associated Splicing Factor; D034741:RNA, Small Interfering / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012171:Retinal Vessels / Q000166:cytology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D019108:Tight Junctions / Q000187:drug effects; D015534:Trans-Activators; D014162:Transfection
chemical_list: C107466:CLDN5 protein, human; D062507:Claudin-5; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D062793:Occludin; D050810:Octamer Transcription Factors; D000072670:PTB-Associated Splicing Factor; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D015534:Trans-Activators; D003907:Dexamethasone
keywords: blood-retinal barrier; enhancer; gene expression; tight junction
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