The Role of α-catenin in stabilization of cell-ECM adhesion

在培养过程中，α-连环蛋白缺陷的 DLD1 细胞从基质上脱离，漂浮在培养基中，然后重新附着到基质上。 α-连环蛋白的表达抑制细胞的脱离。我们进行了以下实验以了解α-连环蛋白的表达导致细胞脱离受到抑制的原因。 1）虽然DNA微阵列分析揭示了一些基质金属蛋白酶基因的差异表达，但实时PCR分析并未证实该结果。 2)添加金属蛋白酶抑制剂并不能抑制细胞的分离。 3)α-catenin表达后整合素或细胞外基质成分的表达没有变化。因此我们认为α-连环蛋白诱导了细胞内部而不是细胞外部的变化。肌动蛋白丝不仅参与细胞形态的维持，还参与细胞运动和生长。 4) 对影响肌动蛋白细胞骨架的试剂进行的分析表明，RhoGTPase 可能参与其中。因此，我们引入了显性负性Rho、组成性活性Rho或显性负性形式的RhoGAP来寻找Rho的作用。然而，构建体的表达并没有抑制细胞的分离。 5)接下来，我们确定了抑制细胞脱离所需的α-连环蛋白区域。我们发现400个氨基酸残基的氨基末端区域或300个氨基酸残基的羧基末端区域是该活性所必需的。沿着这条线的研究目前正在进行中。

During culture, a-catenin-deficient DLD1 cells detach from the substrate, float in the medium, and then reattach to the substratum. Expression of α-catenin suppress the detachment of the cells. We did following experiments in order to know the reason why the expression of α-catenin results in suppression of the cell detachment. 1) Although DNA micro array analysis revealed differential expression of some matrix metalloproteinase genes, real-time PCR analysis, however, did not confirm the results. 2) Addition of inhibitors for the metalloproteinases did not suppress the detachment of the cells. 3) The expression of integrins or the extracellular matrix components did not changed after α-catenin expression. Therefore we thought that the α-catenin induced changes inside of the cells instead of outside of the cells. The actin filaments are involved not only in the maintenance of the cell morphology but also in cell motility and growth. 4) Analysis with reagents that affect actin cytoskeleton suggested possible involvement of RhoGTPase. Therefore we introduced the dominant-negative Rho, the constitutively active Rho, or the dominant-negative form of RhoGAP to find the role of Rho. The expression of the constructs, however, did not suppress the detachment of the cells. 5) Next, we identified the region of α-catenin that is required for the suppression of the cell detachment. We found that the amino-terminal region of 400 amino acid residues or the carboxy-terminal region of 300 amino acid residues is required for the activity. The research along this line is now in progress.