Fujii Naoko, Matsuo Yukinobu, Matsunaga Toshiyuki, Endo Satoshi, Sakai Hideki, Yamaguchi Masahiko, Yamazaki Yasuhiro, Sugatani Junko, Ikari Akira
From the Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196.
the Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, and.
J Biol Chem. 2016 Nov 18;291(47):24787-24799. doi: 10.1074/jbc.M116.728196. Epub 2016 Oct 12.
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.
低渗应激降低了肾小管上皮HK - 2细胞和马-达二氏犬肾细胞中紧密连接蛋白-1和-2的表达水平。在此，我们研究了参与这种降低的调控机制。低渗诱导的紧密连接蛋白表达降低受到以下物质的抑制：SB202190，一种p38丝裂原活化蛋白激酶抑制剂，但不受U0126（一种MEK抑制剂）的抑制；Go6983，一种蛋白激酶C抑制剂；或SP600125，一种Jun氨基末端蛋白激酶抑制剂。低渗应激增加了跨上皮电阻，这被SB202190抑制。低渗应激降低了紧密连接蛋白-1的mRNA表达水平，但未降低紧密连接蛋白-2的mRNA表达水平。低渗应激降低了紧密连接蛋白-1和-2的蛋白质稳定性。低渗诱导的紧密连接蛋白表达降低受到以下物质的抑制：氯喹，一种溶酶体抑制剂；dynasore和单丹磺酰尸胺，网格蛋白依赖性内吞作用抑制剂；以及针对网格蛋白重链的小干扰RNA。在氯喹和单丹磺酰尸胺处理的细胞中，紧密连接蛋白-1和-2分别主要分布在细胞质和紧密连接（TJ）中。低渗应激降低了紧密连接蛋白-1和-2的磷酸化水平，这被蛋白磷酸酶抑制剂冈田酸和斑蝥素抑制。紧密连接蛋白-1和-2的去磷酸化突变体主要分布在细胞质中，在低渗应激下消失。相反，模拟磷酸化突变体分布在紧密连接中，在低渗应激下未减少。我们认为，低渗应激诱导紧密连接蛋白-1和-2去磷酸化、网格蛋白依赖性内吞作用以及在溶酶体中的降解，导致肾小管上皮细胞中紧密连接屏障的破坏。
