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

在培养过程中，缺乏α-连环蛋白的DLD1细胞从基质中分离出来，漂浮在培养基中，然后重新附着在基质上。α-连环蛋白的表达抑制了细胞的脱离。为了了解α-连环蛋白表达导致细胞脱落抑制的原因，我们做了以下实验。1)虽然DNA微阵列分析显示了一些基质金属蛋白酶基因的差异表达，但实时荧光定量PCR分析并不能证实这一结果。2)金属蛋白酶抑制剂的加入不能抑制细胞的脱离。3)α-连环蛋白表达后，整合素或细胞外基质成分的表达无明显变化。因此，我们认为α-连环蛋白是在细胞内而不是在细胞外引起变化。肌动蛋白细丝不仅参与细胞形态的维持，还参与细胞的运动和生长。4)对影响肌动蛋白细胞骨架的试剂的分析表明，RhoGTPase可能参与了这一过程。因此，我们引入了显性-负性Rho、结构性活性Rho或RhoGAP的显性-负性形式来寻找Rho的作用。然而，构建体的表达并没有抑制细胞的脱离。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，活性需要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。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.