title: Hypotonic Stress-induced Down-regulation of Claudin-1 and -2 Mediated by Dephosphorylation and Clathrin-dependent Endocytosis in Renal Tubular Epithelial Cells.
abstract: Hypotonic stress decreased claudin-1 and -2 expression levels in renal tubular epithelial HK-2 and Madin-Darby canine kidney cells. Here, we examined the regulatory mechanism involved in this decrease. The hypotonicity-induced decrease in claudin expression was inhibited by the following: SB202190, a p38 MAPK inhibitor, but not by U0126, a MEK inhibitor; Go6983, a protein kinase C inhibitor; or SP600125, a Jun N-terminal protein kinase inhibitor. Hypotonic stress increased transepithelial electrical resistance, which was inhibited by SB202190. The mRNA expression level of claudin-1 was decreased by hypotonic stress but that of claudin-2 was not. Hypotonic stress decreased the protein stability of claudin-1 and -2. The hypotonicity-induced decrease in claudin expression was inhibited by the following: chloroquine, a lysosome inhibitor; dynasore and monodansylcadaverine, clathrin-dependent endocytosis inhibitors; and siRNA against clathrin heavy chain. Claudin-1 and -2 were mainly distributed in the cytosol and tight junctions (TJs) in the chloroquine- and monodansylcadaverine-treated cells, respectively. Hypotonic stress decreased the phosphorylation levels of claudin-1 and -2, which were inhibited by the protein phosphatase inhibitors okadaic acid and cantharidin. Dephosphorylated mutants of claudin-1 and -2 were mainly distributed in the cytosol, which disappeared in response to hypotonic stress. In contrast, mimicking phosphorylation mutants were distributed in the TJs, which were not decreased by hypotonic stress. We suggest that hypotonic stress induces dephosphorylation, clathrin-dependent endocytosis, and degradation of claudin-1 and -2 in lysosomes, resulting in disruption of the TJ barrier in renal tubular epithelial cells.
authors.affiliation: From the Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196.
authors.forename: Naoko
authors.initials: N
authors.lastname: Fujii
authors.affiliation: From the Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196.
authors.forename: Yukinobu
authors.initials: Y
authors.lastname: Matsuo
authors.affiliation: From the Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Matsunaga
authors.affiliation: From the Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Endo
authors.affiliation: the Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, and.
authors.forename: Hideki
authors.initials: H
authors.lastname: Sakai
authors.affiliation: the School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: the School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Yamazaki
authors.affiliation: the School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Sugatani
authors.affiliation: From the Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196,. Electronic address: ikari@gifu-pu.ac.jp.
authors.forename: Akira
authors.initials: A
authors.lastname: Ikari
mesh_terms: D000818:Animals; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D062445:Claudin-1 / Q000235:genetics / Q000378:metabolism*; D057167:Claudins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D015536:Down-Regulation; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D009997:Osmotic Pressure; D010766:Phosphorylation; D019108:Tight Junctions / Q000235:genetics / Q000378:metabolism
chemical_list: C568108:CLDN1 protein, human; C491875:CLDN2 protein, human; D002966:Clathrin; D062445:Claudin-1; D057167:Claudins
keywords: endocytosis; kidney; lysosome; protein degradation; tight junction