Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
J Cell Physiol. 2011 Sep;226(9):2448-56. doi: 10.1002/jcp.22590.
Epidermal growth factor (EGF) decreases the mRNA and protein levels of claudin-2 (CLDN2) in Madin-Darby canine kidney (MDCK) II cells. Here we examined whether EGF affects the stability and intracellular distribution of CLDN2 protein. EGF decreased surface and total levels of CLDN2, which was inhibited by U0126, a MEK inhibitor. CLDN2 was co-localized at the tight junctions (TJs) with ZO-1, a scaffolding protein. The fluorescence signal for CLDN2 disappeared on treatment with EGF, which was inhibited by U0126. EGF accelerated the decrease in CLDN2 in the presence of cycloheximide, a translation inhibitor, indicating that EGF reduces the stability of the protein. Chloroquine, a lysosomal protease inhibitor, blocked the EGF-induced decrease in CLDN2 protein and caused the co-localization of CLDN2 with Lamp-1, a marker of lysosome. Monodancylcadaverine, an inhibitor of endocytosis, and clathrin siRNA blocked the EGF-induced decrease in CLDN2 and the translocation of CLDN2 from the TJs to the lysosome. EGF increased the association of CLDN2 with clathrin and adaptin α which was inhibited by U0126. These results suggest that EGF accelerates clathrin-dependent endocytosis and lysosomal degradation of CLDN2 protein mediated by the activation of a MEK/ERK pathway.
表皮生长因子（EGF）降低 Madin-Darby 犬肾（MDCK）II 细胞中 Claudin-2（CLDN2）的 mRNA 和蛋白水平。在这里，我们研究了 EGF 是否会影响 CLDN2 蛋白的稳定性和细胞内分布。EGF 降低了 CLDN2 的表面和总水平，而 MEK 抑制剂 U0126 则抑制了这一过程。CLDN2 与紧密连接（TJ）上的 ZO-1 共定位，后者是一种支架蛋白。用 EGF 处理后，CLDN2 的荧光信号消失，而 U0126 则抑制了这一过程。在翻译抑制剂环己酰亚胺存在的情况下，EGF 加速了 CLDN2 的减少，表明 EGF 降低了蛋白质的稳定性。溶酶体蛋白酶抑制剂氯喹阻断了 EGF 诱导的 CLDN2 蛋白减少，并导致 CLDN2 与溶酶体标记物 Lamp-1 共定位。内吞作用抑制剂单丹酰cadaverine 和网格蛋白 siRNA 阻断了 EGF 诱导的 CLDN2 减少和 CLDN2 从 TJ 向溶酶体的易位。EGF 增加了 CLDN2 与网格蛋白和衔接蛋白α的结合，而 U0126 则抑制了这种结合。这些结果表明，EGF 通过激活 MEK/ERK 通路，加速了 CLDN2 蛋白的网格蛋白依赖性内吞作用和溶酶体降解。
