Hernandez-Blazquez F J, Joazeiro P P, Omori Y, Yamasaki H
Unit of Multistage Carcinogenesis, International Agency for Research on Cancer, 150 cours Albert Thomas, Lyon Cedex 08, 69372, France.
Exp Cell Res. 2001 Nov 1;270(2):235-47. doi: 10.1006/excr.2001.5342.
The gap junctional intercellular communication-deficient mouse skin papilloma cell line P3/22 expresses Cx43 but not E-cadherin. The E-cadherin gene-transfected cells (P3E1) communicate in a calcium-dependent manner and they were used to study how E-cadherin restores the function of connexins. At low calcium, Cx43 molecules remain in the cytoplasm of P3E1 cells and appear at cell-cell contact areas only in high-calcium medium. While Cx43 is unphosphorylated in P3E1 cells in low-calcium medium, two phosphorylated bands appeared at high calcium. However, when Cx26, which has no C-terminal tail that can undergo phosphorylation, was expressed in P3E1 cells, this connexin also moved to the plasma membrane after the calcium shift and partly colocalized with Cx43, suggesting that C-terminal phosphorylation is not essential for E-cadherin-mediated intracellular transport of connexins. In low calcium, both Cx26 and Cx43 remained and colocalized in the endoplasmic reticulum. As early as 30 min after the shift to high-calcium medium, both Cx43 and Cx26 began to accumulate in the Golgi apparatus. Intracellular movement of connexins to the cytoplasmic membrane at high calcium was effectively blocked by cytochalasin D and brefeldin A. These results suggest that E-cadherin junction formation at high calcium leads to formation of actin cables, which directly or indirectly transport connexins from the cytoplasm to the cell-cell contact membranes via the Golgi apparatus.
缝隙连接细胞间通讯缺陷的小鼠皮肤乳头瘤细胞系P3/22表达Cx43，但不表达E-钙黏蛋白。转染了E-钙黏蛋白基因的细胞（P3E1）以钙依赖的方式进行通讯，它们被用于研究E-钙黏蛋白如何恢复连接蛋白的功能。在低钙条件下，Cx43分子保留在P3E1细胞的细胞质中，仅在高钙培养基中出现在细胞-细胞接触区域。虽然在低钙培养基中P3E1细胞中的Cx43未被磷酸化，但在高钙条件下出现了两条磷酸化条带。然而，当没有可进行磷酸化的C末端尾巴的Cx26在P3E1细胞中表达时，这种连接蛋白在钙转移后也移动到质膜，并与Cx43部分共定位，这表明C末端磷酸化对于E-钙黏蛋白介导的连接蛋白细胞内转运不是必需的。在低钙条件下，Cx26和Cx43都保留在内质网中并共定位。早在转移到高钙培养基30分钟后，Cx43和Cx26就开始在高尔基体中积累。细胞松弛素D和布雷菲德菌素A有效地阻断了连接蛋白在高钙条件下向细胞质膜的细胞内移动。这些结果表明，高钙条件下E-钙黏蛋白连接的形成导致肌动蛋白丝的形成，其直接或间接通过高尔基体将连接蛋白从细胞质转运到细胞-细胞接触膜。
