Glucocorticoid induction of occludin expression and endothelial barrier requires transcription factor p54 NONO

Abstract

Purpose: 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.

Keywords: blood-retinal barrier; enhancer; gene expression; tight junction.

Figure 1

The OEE binds the transcription factors p54/NONO and PSF following dexamethasone treatment. (A) Nuclear extract was prepared from dexamethasone-treated BREC and incubated with ³²P-labeled OEE oligos. EMSA analysis showed selective binding of a protein complex to the labeled probe, which was competed away by addition of an excess of cold probe. The scrambled control does not bind the complex. (B) HRECs were treated with dexamethasone for 24 hours and nuclear extract (NE) was incubated with Dynabead-immobilized OEE oligos in a DNA affinity purification protocol. Flow-through (FT), successive washes (W1–W4) and a high salt elution (E) were run on SDS-PAGE to identify bound complexes, demonstrating selective enrichment of certain proteins. Bands were excised from the gel and analyzed by MS/MS following trypsin digest. The bands identified as p54 and PSF are indicated. Confirmation of the MS/MS analysis is shown in (C), as p54 and PSF from dexamethasone-treated nuclear extract bound the immobilized OEE strongly following DNA affinity purification followed by Western blot.

Figure 2

p54 and PSF bind to the OEE in cells and retina. (A) HRECs, under basal conditions and after dexamethasone treatment, were treated with formaldehyde to cross-link DNA and trans-acting factors. ChIP assays showed the binding of p54 and PSF to the OEE or homologous OEE present in the promoters of occludin, claudin-5, and cadherin-9 using specific primers flanking the OEE sequence. The distal region of the claudin-5 promoter region not containing the OEE sequence served as a negative control. (B) Rats were killed and retinas were flash frozen. The tissue was subjected to the same ChIP protocol to show that p54 and PSF basally bound the OEE in vivo in rodent retina.

Figure 3

p54 knockdown ablates the steroid-induction of occludin and claudin-5. (A) HRECs were transfected with siRNA targeted against p54 and treated with or without dexamethasone in stepdown media. The GC-induced increase of occludin and claudin-5 is prevented by knockdown of p54 using a smartpool of multiple p54-targeted siRNA oligos and individually targeted oligos (#10 and #11). (B) Quantification of protein levels by Western blot showed a significant ablation of dexamethasone-induction of both occludin and claudin-5. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

p54 knockdown prevents the barrier induction properties of dexamethasone. (A) BRECs were transfected with siRNA targeting p54 and seeded on 0.4-μm Transwell filters. After 48 hours, cells were switched to stepdown media with or without dexamethasone for 24 hours. Permeability to 70-kDa RITC-dextran tracer over a 4-hour time period was measured. The reduction in endothelial permeability to the dextran after dexamethasone treatment was ablated by p54 knockdown (P < 0.05). (B) BRECs transfected with siRNA against p54 were seeded on 8W10E+ arrays and TER was measured continually on the ECIS Z-theta instrument. GC treatment increased electrical resistance and knockdown of p54 decreased the GC induction of electrical resistance. After 13 hours of dexamethasone treatment and until the end of the experiment, p54 knockdown significantly reduced the glucocorticoid-induction compared with control (P < 0.001). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Known cis-regulatory sequences of the occludin and claudin-5 promoters. A schematic of occludin and claudin-5 promoters is presented showing activating cis-sequences above the line and inhibitory cis-sequences below the line. (A) The occludin promoter is shown from −2000 to the transcriptional start site, indicating clustered inhibitory YY1 sites, an imperfect distal GRE, clustered SP3 sites near the transcription start site, lung oncogene TTF-1, and the RH4 of the OEE, −126 to −114 of the promoter. (B) The claudin-5 promoter is positively regulated by FoxO1, ERG, and SOX18, and inhibited by nuclear factor–κB signaling. The homologous OEE is shown at position −1159 to −1147 of the promoter.