Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells
Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells
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DOI：
10.1152/ajpcell.00027.2008
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发表时间：
2008-06-01
影响因子：
5.5
通讯作者：
Basson, Marc D.
中科院分区：
文献类型：
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作者：
Chaturvedi, Lakshmi S.;Gayer, Christopher P.;Basson, Marc D.
由于绒毛运动或蠕动引起的重复变形可能支持肠粘膜，在正常情况下刺激肠上皮细胞增殖，并在富含组织纤连蛋白的损伤和发炎粘膜中恢复。周期性应变增强Caco-2和IEC-6肠上皮细胞通过ERK跨纤连蛋白迁移。然而，ERK激活的上游介质是未知的。我们研究了Src和FAK是否介导了人Caco-2肠上皮细胞在纤连蛋白上应变诱导的ERK磷酸化和迁移。组织纤维连接蛋白预包被膜上的单层以10个循环/分钟进行平均10%的重复变形。变形显著增加了Src-Tyr（418）、FAK-Tyr（397）-Tyr（576）-Tyr（925）和ERK的磷酸化。用PP 2（10 μ mol/l）或特异性短干扰（si）RNA阻断Src，可防止菌株刺激伤口闭合。Src抑制也阻止了菌株诱导的FAK在Tyr（397）和Tyr（576）的磷酸化，但不能阻止FAK-Tyr（925）或ERK磷酸化。通过siRNA减少FAK抑制应变诱导的ERK磷酸化。转染NH 2端酪氨酸磷酸化缺陷型FAK突变体Y397 F、Y 576 F-Y 577 F和Y397 F-Y 576 F-Y 577 F不能阻止环状菌株对ERK 2的激活，但COOH端的FAK突变体（Y 925 F）阻止了菌株诱导的ERK 2激活。尽管Y397 F-Y 576 F-Y 577 F FAK构建体在静态条件下表现出较少的基础FAK-Tyr（925）磷酸化，但其仍然表现出响应于菌株的增加的FAK-Tyr（925）磷酸化。这些结果表明，重复变形刺激肠上皮细胞运动的方式，需要Src激活和一种新的Src独立FAK-Tyr（925）依赖性途径，激活ERK的纤连蛋白。该途径可能是在肠梗阻或禁食的情况下促进粘膜愈合的干预措施的重要靶点。
Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-Tyr(418), FAK-Tyr(397)-Tyr(576)-Tyr(925), and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 mu mol/l) or specific short interfering ( si) RNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr(397) and Tyr(576) but not FAK-Tyr(925) or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of NH2-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK-Tyr(925) phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr(925) phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr(925)-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting.