Okamoto Ryoko, Irie Kenji, Yamada Akio, Katata Tatsuo, Fukuhara Atsunori, Takai Yoshimi
Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.
Genes Cells. 2005 May;10(5):435-45. doi: 10.1111/j.1365-2443.2005.00846.x.
The formation of tight junctions (TJs) is dependent on the formation of adherens junctions (AJs) in MDCK cells. E-Cadherin and nectin are major cell-cell adhesion molecules (CAMs) at AJs, whereas claudin, occludin and junctional adhesion molecule (JAM) are major CAMs at TJs. When MDCK cells precultured at 2 microm Ca(2+) are cultured at 2 mm Ca(2+), nectin first forms cell-cell adhesion and recruits E-cadherin to the nectin-based cell-cell adhesion sites to form AJs. Thereafter, nectin recruits first JAM-A and then claudin-1 and occludin to the apical side of AJs to form TJs. In contrast, when MDCK cells precultured at 2 microm Ca(2+) are cultured at 2 microm Ca(2+) in the presence of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a TJ-like structure is formed without the formation of the E-cadherin-based AJs. We showed here that GFP-E-cadherin, which did not trans-interact due to 2 microm Ca(2+) but associated with alpha- and beta-catenins and p120(ctn), was recruited to the nectin-based cell-cell adhesion sites by the action of TPA. The nectin inhibitors, which inhibited the trans-interaction of nectin, inhibited the recruitment of GFP-E-cadherin and their associating catenins by the action of TPA. Microbeads coated with the extracellular fragment of nectin recruited not only cellular nectin but also GFP-E-cadherin and their associating catenins by the action of TPA. These results indicate that when the TJ-like structure is formed by the action of TPA, non-trans-interacting E-cadherin and its associating catenins are recruited to the nectin-based cell-cell adhesion sites and that the trans-interaction of E-cadherin is not essential for the formation of TJs.
紧密连接（TJ）的形成依赖于MDCK细胞中黏附连接（AJ）的形成。E-钙黏蛋白和nectin是AJ处主要的细胞间黏附分子（CAM），而闭合蛋白、闭锁蛋白和连接黏附分子（JAM）是TJ处主要的CAM。当在2 mM钙离子浓度下培养预培养于2 μM钙离子浓度的MDCK细胞时，nectin首先形成细胞间黏附，并将E-钙黏蛋白募集到基于nectin的细胞间黏附位点以形成AJ。此后，nectin首先将JAM-A然后将闭合蛋白-1和闭锁蛋白募集到AJ的顶端侧以形成TJ。相反，当在佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）存在下，将预培养于2 μM钙离子浓度的MDCK细胞在2 μM钙离子浓度下培养时，会形成类似TJ的结构，而不会形成基于E-钙黏蛋白的AJ。我们在此表明，由于2 μM钙离子浓度而不发生反式相互作用但与α-和β-连环蛋白以及p120（ctn）相关联的GFP-E-钙黏蛋白，通过TPA的作用被募集到基于nectin的细胞间黏附位点。抑制nectin反式相互作用的nectin抑制剂，通过TPA的作用抑制了GFP-E-钙黏蛋白及其相关连环蛋白的募集。涂有nectin细胞外片段的微珠通过TPA的作用不仅募集了细胞内的nectin，还募集了GFP-E-钙黏蛋白及其相关连环蛋白。这些结果表明，当通过TPA的作用形成类似TJ的结构时，非反式相互作用的E-钙黏蛋白及其相关连环蛋白被募集到基于nectin的细胞间黏附位点，并且E-钙黏蛋白的反式相互作用对于TJ的形成不是必需的。
