Liu Y W, Sanders M A, Basson M D
Department of Surgery, Yale University, New Haven, Connecticut, 06520, USA.
J Surg Res. 1998 Jul 1;77(2):112-8. doi: 10.1006/jsre.1998.5369.
Intestinal epithelial cells assume a specialized phenotype adapted to motility and mucosal healing during mucosal restitution. Since cell-matrix interactions initiate tyrosine kinase (TK) signals, we hypothesized that the regulation of the intestinal epithelial migratory phenotype may also involve TK signals, particularly via focal adhesion kinase (FAK). Caco-2 cells were seeded simultaneously at 26,000 and 6000 cells/cm2. After 4 days, the first cells were confluent, while cells in the second population were not contact-inhibited and expressed migrating lamellipodia. Cells were fractionated into Triton X-100-soluble (membrane/cytoskeletal) and -insoluble (cytosolic) fractions. TK activity in each fraction was assayed by ELISA using a synthetic substrate. FAK protein was assessed by immunoprecipitation with monoclonal anti-FAK and Western blotting. Because active FAK autophosphorylates, we also measured FAK tyrosine phosphorylation, immunoprecipitating with anti-FAK and then Western blotting for phosphotyrosine. TK activity was increased in both cytosolic and membrane/cytoskeletal fractions of migrating cells by 17.6 +/- 3.6 and 28.9 +/- 4.1%, respectively, compared to static cells (n = 11, P < 0.01). FAK protein increased in the cytosolic fraction by 90.4 +/- 20.0% (n = 5, P = 0.01), but did not change in the membrane/cytoskeletal fraction. Tyrosine phosphorylated FAK increased by 62.8 +/- 21.4% in the cytosolic fraction of migrating cells but also by 46.6 +/- 38.4% in the membrane/cytoskeletal fraction (n = 5, P < 0.05). These data suggest that intestinal epithelial cell migration is associated with increases in both cytosolic and cytoskeletal TK activity and upregulation of cytosolic FAK protein. The increase in cytoskeletal FAK phosphorylation without increased FAK protein suggests autophosphorylation and increased cytoskeletal FAK activity. The migrating intestinal epithelial phenotype may therefore be modulated by TK signals including cytoskeletal FAK activity.
在黏膜修复过程中，肠上皮细胞呈现出一种适应运动和黏膜愈合的特殊表型。由于细胞与基质的相互作用会启动酪氨酸激酶（TK）信号，我们推测肠上皮迁移表型的调节可能也涉及TK信号，尤其是通过粘着斑激酶（FAK）。将Caco-2细胞分别以26,000个细胞/cm²和6000个细胞/cm²的密度同时接种。4天后，第一批细胞汇合，而第二批细胞未受到接触抑制，并表现出迁移的片状伪足。将细胞分为Triton X-100可溶性（膜/细胞骨架）和不可溶性（胞质）部分。使用合成底物通过酶联免疫吸附测定法（ELISA）检测各部分的TK活性。通过用单克隆抗FAK进行免疫沉淀和蛋白质印迹法评估FAK蛋白。由于活性FAK会发生自身磷酸化，我们还通过用抗FAK进行免疫沉淀然后对磷酸酪氨酸进行蛋白质印迹法来测量FAK酪氨酸磷酸化。与静止细胞相比，迁移细胞的胞质和膜/细胞骨架部分的TK活性分别增加了17.6±3.6%和28.9±4.1%（n = 11，P < 0.）。FAK蛋白在胞质部分增加了90.4±20.0%（n = 5，P = 0.01），但在膜/细胞骨架部分没有变化。迁移细胞的胞质部分酪氨酸磷酸化的FAK增加了62.8±21.4%，但在膜/细胞骨架部分也增加了46.6±38.4%（n = 5，P < 0.05）。这些数据表明，肠上皮细胞迁移与胞质和细胞骨架TK活性的增加以及胞质FAK蛋白的上调有关。细胞骨架FAK磷酸化增加而FAK蛋白未增加表明发生了自身磷酸化并增加了细胞骨架FAK活性。因此，迁移的肠上皮表型可能受包括细胞骨架FAK活性在内的TK信号调节。
