title: Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: involvement of NFAT5.
abstract: OBJECTIVE
  High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration. Neovascular retinal diseases are associated with edema. Various factors and channels, including vascular endothelial growth factor (VEGF) and aquaporins (AQPs), influence neovascularization and the development of edema. Therefore, we determined whether extracellular hyperosmolarity alters the expression of VEGF and AQPs in cultured human retinal pigment epithelial (RPE) cells.
  
  
  METHODS
  Human RPE cells obtained within 48 h of donor death were prepared and cultured. Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. The levels of signaling proteins and nuclear factor of activated T cell 5 (NFAT5) were determined by western blotting. DNA binding of NFAT5 was determined with EMSA. NFAT5 was knocked down with siRNA.
  
  
  RESULTS
  Extracellular hyperosmolarity stimulated VEGF gene transcription and the secretion of VEGF protein. Hyperosmolarity also increased the gene expression of AQP5 and AQP8, induced the phosphorylation of p38 MAPK and ERK1/2, increased the expression of HIF-1α and NFAT5, and induced the DNA binding of NFAT5. The hyperosmotic expression of VEGF was dependent on the activation of p38 MAPK, ERK1/2, JNK, PI3K, HIF-1, and NFAT5. The hyperosmotic induction of AQP5 was in part dependent on the activation of p38 MAPK, ERK1/2, NF-κB, and NFAT5. Triamcinolone acetonide inhibited the hyperosmotic expression of VEGF but not AQP5. The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia.
  
  
  CONCLUSIONS
  Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells. The data suggest that high salt intake resulting in osmotic stress may aggravate neovascular retinal diseases and edema via the stimulation of VEGF production in RPE. The downregulation of AQP5 under hypoxic conditions may prevent the resolution of edema.
authors: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors: Margrit
authors: M
authors: Hollborn
authors: Paul Flechsig Institute of Brain Research, University of Leipzig, Leipzig, Germany.
authors: Stefanie
authors: S
authors: Vogler
authors: Paul Flechsig Institute of Brain Research, University of Leipzig, Leipzig, Germany.
authors: Andreas
authors: A
authors: Reichenbach
authors: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors: Peter
authors: P
authors: Wiedemann
authors: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.
authors: Andreas
authors: A
authors: Bringmann
authors: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany ; Helios Klinikum Aue, Aue, Germany.
authors: Leon
authors: L
authors: Kohen
mesh_terms: D051402:Aquaporin 5 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D010766:Phosphorylation / Q000187:drug effects; D061251:Primary Cell Culture; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction; D012965:Sodium Chloride / Q000494:pharmacology*; D013395:Sucrose / Q000494:pharmacology; D014157:Transcription Factors / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014222:Triamcinolone Acetonide / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: C496036:AQP5 protein, human; D051402:Aquaporin 5; D020346:Aquaporins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; C118125:NFAT5 protein, human; D034741:RNA, Small Interfering; D014157:Transcription Factors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C108330:aquaporin 8; D012965:Sodium Chloride; D013395:Sucrose; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D014222:Triamcinolone Acetonide