Dept. of Surgery, Michigan State University, East Lansing, MI 48912, USA.
Am J Physiol Cell Physiol. 2011 Feb;300(2):C356-67. doi: 10.1152/ajpcell.00273.2010. Epub 2010 Nov 17.
The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. Such repetitive strain promotes intestinal epithelial migration across fibronectin in vitro, but signaling mediators for this are poorly understood. We hypothesized that integrin-linked kinase (ILK) mediates strain-stimulated migration in intestinal epithelial cells cultured on fibronectin. ILK kinase activity increased rapidly 5 min after strain induction in both Caco-2 and intestinal epithelial cell-6 (IEC-6) cells. Wound closure in response to strain was reduced in ILK small interfering RNA (siRNA)-transfected Caco-2 cell monolayers when compared with control siRNA-transfected Caco-2 cells. Pharmacological blockade of phosphatidylinositol-3 kinase (PI3K) or Src or reducing Src by siRNA prevented strain activation of ILK. ILK coimmunoprecipitated with focal adhesion kinase (FAK), and this association was decreased by mutation of FAK Tyr925 but not FAK Tyr397. Strain induction of FAK Tyr925 phosphorylation but not FAK Tyr397 or FAK Tyr576 phosphorylation was blocked in ILK siRNA-transfected cells. ILK-Src association was stimulated by strain and was blocked by the Src inhibitor PP2. Finally, ILK reduction by siRNA inhibited strain-induced phosphorylation of myosin light chain and Akt. These results suggest a strain-dependent signaling pathway in which ILK association with FAK and Src mediates the subsequent downstream strain-induced motogenic response and suggest that ILK induction by repetitive deformation may contribute to recovery from mucosal injury and restoration of the mucosal barrier in patients with prolonged ileus. ILK may therefore be an important target for intervention to maintain the mucosa in such patients.
肠上皮在正常肠道功能中通过蠕动和绒毛运动反复受到变形。这种反复的张力促进了肠上皮细胞在体外跨越纤维连接蛋白的迁移，但对于这种迁移的信号介质知之甚少。我们假设整合素连接激酶（ILK）介导了培养在纤维连接蛋白上的肠上皮细胞中应变刺激的迁移。在 Caco-2 和肠上皮细胞-6（IEC-6）细胞中，应变诱导后 5 分钟内，ILK 激酶活性迅速增加。与对照 siRNA 转染的 Caco-2 细胞单层相比，ILK 小干扰 RNA（siRNA）转染的 Caco-2 细胞单层中的伤口闭合对应变的反应减少。PI3K 或Src 的药理学阻断或通过 siRNA 减少 Src 可防止应变激活 ILK。ILK 与粘着斑激酶（FAK）共免疫沉淀，并且这种关联通过 FAK Tyr925 的突变而减少，但不通过 FAK Tyr397 或 FAK Tyr576 的突变。在 ILK siRNA 转染的细胞中，应变诱导的 FAK Tyr925 磷酸化而不是 FAK Tyr397 或 FAK Tyr576 磷酸化被阻断。应变诱导的 FAK Tyr397 磷酸化和 FAK Tyr576 磷酸化在 ILK siRNA 转染的细胞中未被阻断。ILK-Src 关联被应变刺激，并且被 Src 抑制剂 PP2 阻断。最后，ILK 的减少通过 siRNA 抑制了应变诱导的肌球蛋白轻链和 Akt 的磷酸化。这些结果表明，在依赖应变的信号通路中，ILK 与 FAK 和 Src 的关联介导了随后的应变诱导的促迁移反应，并且提示重复变形诱导的 ILK 可能有助于从粘膜损伤中恢复并恢复长期肠梗阻患者的粘膜屏障。ILK 因此可能是维持此类患者粘膜的重要干预靶点。
