title: Hyperosmolarity-Induced Down-Regulation of Claudin-2 Mediated by Decrease in PKCβ-Dependent GATA-2 in MDCK Cells.
abstract: Hyperosmolarity decreases claudin-2 expression in renal tubular epithelial cells, but the molecular mechanism remains undefined. Here, we found that the hyperosmolarity-induced decrease in claudin-2 expression is inhibited by Go6983, a non-selective protein kinase C (PKC) inhibitor, and PKCβ specific inhibitor in Madin-Darby canine kidney II cells. Hyperosmolarity increased intracellular free Ca(2+) concentration and phosphorylated PKCβ level, which were inhibited by RN-1734, an antagonist of transient receptor potential vanilloid 4 channel. Phorbol 12-myristate 13-acetate, a PKC activator, decreased claudin-2 expression. These results indicate hyperosmolarity decreases claudin-2 expression mediated by the activation of RN-1734-sensitive channel and PKCβ. Hyperosmolarity decreased promoter activity of claudin-2, which was inhibited by Go6983 and PKCβ inhibitor similar to those in real-time PCR and Western blotting. The effect of hyperosmolarity on promoter activity was not observed in the construct of -469/-6, a deletion mutant. Claudin-2 has hyperosmolarity-sensitive region in its promoter, which includes GATA binding site. Hyperosmolarity decreased the nuclear level of GATA-2, which was inhibited by Go6983 and PKCβ inhibitor. Mutation of GATA binding site decreased the basal promoter activity and inhibited the effect of hyperosmolarity. In contrast, the hyperosmolarity-induced decrease in reporter activity and claudin-2 expression were rescued by over-expression of wild type GATA-2. Chromatin immunoprecipitation assay showed that GATA-2 bound to promoter region of claudin-2. These results suggest that hyperosmolarity decreases the expression level of claudin-2 via a decrease in PKCβ-dependent GATA-2 transcriptional activity in renal tubular epithelial cells.
authors.affiliation: Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, Japan.|School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Ikari
authors.affiliation: Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Fujii
authors.affiliation: School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Hahakabe
authors.affiliation: School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Hisayoshi
authors.initials: H
authors.lastname: Hayashi
authors.affiliation: School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Yamazaki
authors.affiliation: Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Endo
authors.affiliation: Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Matsunaga
authors.affiliation: School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Sugatani
mesh_terms: D000818:Animals; D001665:Binding Sites; D020013:Calcium Signaling / Q000187:drug effects; D062446:Claudin-2 / Q000096:biosynthesis* / Q000235:genetics; D004285:Dogs; D050989:GATA2 Transcription Factor / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D007211:Indoles / Q000008:administration & dosage; D007687:Kidney Tubules, Proximal / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D008301:Maleimides / Q000008:administration & dosage; D009994:Osmolar Concentration; D011401:Promoter Regions, Genetic; D064546:Protein Kinase C beta / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D051381:Rats; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate / Q000008:administration & dosage
chemical_list: C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D062446:Claudin-2; D050989:GATA2 Transcription Factor; D007211:Indoles; D008301:Maleimides; C544743:RN 1734; D013449:Sulfonamides; D064546:Protein Kinase C beta; D013755:Tetradecanoylphorbol Acetate