title: Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress.
abstract: OBJECTIVE
  Macular edema, a frequently encountered complication of diabetic retinopathy (DR), results from alterations of the blood retinal barrier (BRB) and leads to modifications of the retinal pigmented epithelium (RPE) functions. Osmolar changes of the surrounding medium could be responsible for modifications of the RPE functions leading to disturbance of retinal homeostasis. The expression, activation and function of the key hyperosmolar response factor Tonicity Enhancer Binding Protein (TonEBP also called nuclear factor of activated T-cell 5 - NFTA5) was investigated in ARPE-19 cells, derived from human RPE, in response to hyperosmolar stimulation.
  
  
  METHODS
  ARPE-19 cells were exposed to hyperosmolar medium. TonEBP mRNA and protein levels were quantified by qRT-PCR and semi-quantitative Western blot. TonEBP nuclear translocation was investigated by immunofluorescence. TonEBP transactivation activity was measured using a reported plasmid containing TonEBP binding sites.
  
  
  RESULTS
  In response to hyperosmolar stimulation of ARPE-19 cells, a dose-dependent increase in TonEBP mRNA and protein levels, as well as TonEBP nuclear translocation were observed. TonEBP transactivation activity was further demonstrated using a reporter plasmid containing TonEBP binding sites. A dominant negative form of TonEBP abolished NaCl-induced increase in TonEBP transactivation activity, and inhibited the increase of the target genes aldose reductase and sodium-dependent taurine transporter mRNA levels. SB203580, an inhibitor of two of the p38 protein kinase's isoforms (p38α and p38β) inhibited the TonEBP nuclear translocation and transactivation activity in ARPE-19 cells exposed to hyperosmolar stimulation.
  
  
  CONCLUSIONS
  Our data demonstrates the involvement of TonEBP in the mechanisms responsible for osmoadaptation to hyperosmolar stress in RPE cells. Given the emerging role of TonEBP in different pathological pathways, these data open new perspectives for the analysis of the mechanisms involved in the modification of functions of the RPE during macular edema.
authors.affiliation: Laboratory of Pathophysiological and Nutritional Biochemistry, Erasme, Brussels, Belgium; Department of Ophthalmology, CHU Saint-Pierre and Brugmann, Brussels, Belgium.
authors.forename: Sarah
authors.initials: S
authors.lastname: Libert
authors.affiliation: I.R.I.B.H.M., campus Erasme, Brussels, Belgium; Department of Ophthalmology, CHU Saint-Pierre and Brugmann, Brussels, Belgium.
authors.forename: François
authors.initials: F
authors.lastname: Willermain
authors.affiliation: Laboratory of Pathophysiological and Nutritional Biochemistry, Erasme, Brussels, Belgium.
authors.forename: Célia
authors.initials: C
authors.lastname: Weber
authors.affiliation: Laboratory of Pathophysiological and Nutritional Biochemistry, Erasme, Brussels, Belgium.
authors.forename: Angélic
authors.initials: A
authors.lastname: Bryla
authors.affiliation: Laboratory of Pathophysiological and Nutritional Biochemistry, Erasme, Brussels, Belgium; Department of Ophthalmology, CHU Saint-Pierre and Brugmann, Brussels, Belgium.
authors.forename: Dany
authors.initials: D
authors.lastname: Salik
authors.affiliation: Laboratory of Pathophysiological and Nutritional Biochemistry, Erasme, Brussels, Belgium.
authors.forename: Françoise
authors.initials: F
authors.lastname: Gregoire
authors.affiliation: Laboratory of Pathophysiological and Nutritional Biochemistry, Erasme, Brussels, Belgium.
authors.forename: Nargis
authors.initials: N
authors.lastname: Bolaky
authors.affiliation: Department of Ophthalmology, CHU Saint-Pierre and Brugmann, Brussels, Belgium.
authors.forename: Laure
authors.initials: L
authors.lastname: Caspers
authors.affiliation: Laboratory of Pathophysiological and Nutritional Biochemistry, Erasme, Brussels, Belgium.
authors.forename: Jason
authors.initials: J
authors.lastname: Perret
authors.affiliation: Laboratory of Pathophysiological and Nutritional Biochemistry, Erasme, Brussels, Belgium.
authors.forename: Christine
authors.initials: C
authors.lastname: Delporte
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D007093:Imidazoles; C118125:NFAT5 protein, human; D011725:Pyridines; D012333:RNA, Messenger; D014157:Transcription Factors; D012965:Sodium Chloride; C093642:SB 203580