Delineating the signals by which repetitive deformation stimulates intestinal epithelial migration across fibronectin
Delineating the signals by which repetitive deformation stimulates intestinal epithelial migration across fibronectin
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DOI：
10.1152/ajpgi.90648.2008
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发表时间：
2009-04-01
影响因子：
4.5
通讯作者：
Basson, Marc D.
中科院分区：
文献类型：
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作者：
Gayer, Christopher P.;Chaturvedi, Lakshmi S.;Basson, Marc D.
重复应变刺激肠上皮细胞迁移通过黏着斑激酶（FAK），Src，和细胞外信号相关激酶（ERK），虽然这些信号的作用和相互作用仍不清楚。我们假设PI 3 K是这一通路的核心。我们将Caco-2和肠上皮细胞-6细胞在柔性纤连蛋白包被的膜上进行10个周期/分钟的变形，通过伤口闭合测定迁移，并通过免疫印迹测定信号传导。菌株刺激PI 3 K、AKT、糖原合成酶激酶（GSK）和p38磷酸化。阻断每个激酶防止应变刺激迁移。阻断PI 3 K阻止了应变刺激的ERK和p38磷酸化。阻止AKT没有。在下游，阻断PI 3 K、AKT或ERK可抑制菌株诱导的GSK-Ser 9磷酸化。在AKT的上游，通过siRNA减少FAK或Rac 1阻断菌株刺激的AKT磷酸化，但通过PP 2或siRNA抑制Src没有。用Tyr 397、Tyr 576/577或Tyr 925处的FAK点突变体转染证明，菌株刺激的AKT磷酸化仅需要FAK 925磷酸化。肌球蛋白轻链激活应变需要FAK，Rac 1，PI 3 K，AKT，GSK和ERK，但不是Src或p38。最后，blebbistatin，一种非肌肉肌球蛋白II抑制剂，阻断了肌球蛋白轻链下游菌株的运动效应。因此，菌株通过包括Src、FAK、Rac 1、PI 3 K、AKT、GSK、ERK、p38、肌球蛋白轻链和肌球蛋白II的复杂途径刺激肠上皮细胞迁移穿过纤连蛋白。
Repetitive strain stimulates intestinal epithelial migration across fibronectin via focal adhesion kinase (FAK), Src, and extracellular signal-related kinase (ERK) although how these signals act and interact remains unclear. We hypothesized that PI3K is central to this pathway. We subjected Caco-2 and intestinal epithelial cell-6 cells to 10 cycles/min deformation on flexible fibronectin-coated membranes, assayed migration by wound closure, and signaling by immunoblots. Strain stimulated PI3K, AKT, glycogen synthase kinase (GSK), and p38 phosphorylation. Blocking each kinase prevented strain stimulation of migration. Blocking PI3K prevented strain-stimulated ERK and p38 phosphorylation. Blocking AKT did not. Downstream, blocking PI3K, AKT, or ERK inhibited strain-induced GSK-Ser9 phosphorylation. Upstream of AKT, reducing FAK or Rac1 by siRNA blocked strain-stimulated AKT phosphorylation, but inhibiting Src by PP2 or siRNA did not. Transfection with FAK point mutants at Tyr397, Tyr576/577, or Tyr925 demonstrated that only FAK925 phosphorylation is required for strain-stimulated AKT phosphorylation. Myosin light chain activation by strain required FAK, Rac1, PI3K, AKT, GSK, and ERK but not Src or p38. Finally, blebbistatin, a nonmuscle myosin II inhibitor, blocked the motogenic effect of strain downstream of myosin light chain. Thus strain stimulates intestinal epithelial migration across fibronectin by a complex pathway including Src, FAK, Rac1, PI3K, AKT, GSK, ERK, p38, myosin light chain, and myosin II.