Requirement of Ca2+ influx- and phosphatidylinositol 3-kinase-mediated m-calpain activity for shear stress-induced endothelial cell polarity
Requirement of Ca2+ influx- and phosphatidylinositol 3-kinase-mediated m-calpain activity for shear stress-induced endothelial cell polarity
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DOI：
10.1152/ajpcell.00083.2007
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发表时间：
2007-10-01
影响因子：
5.5
通讯作者：
Ohata, Hisayuki
中科院分区：
文献类型：
--
作者：
Miyazaki, Takuro;Honda, Kazuo;Ohata, Hisayuki
使用荧光底物和激光扫描共聚焦显微镜测量剪切的人脐静脉内皮细胞 (HUVEC) 的蛋白水解活性，以阐明细胞内 Ca2+ 敏感蛋白酶钙蛋白酶在这些细胞中响应剪切应力的关键作用。在生理剪切范围内，细胞的活性以剪切依赖性方式增强。短干扰RNA诱导的m-钙蛋白酶沉默抑制了活性，但mu-钙蛋白酶没有沉默。去除细胞外 Ca2+ 或应用细胞内 Ca2+ 螯合剂 (BAPTA/AM) 或非选择性阳离子通道阻断剂 (Gd3+) 都会降低蛋白水解活性。此外，磷脂酰肌醇二磷酸（PIP2）螯合剂（新霉素）或磷脂酰肌醇3-激酶（PI3K）抑制剂（LY294002）可抑制活性；相反，被 ERK 激酶抑制剂（U0126、PD98059）部分抑制的活性不受 PLC 抑制剂（U73122）的影响。此外，由剪切引起的 PI3K 下游的 Akt 磷酸化可被新霉素减弱，但不会被钙蛋白酶抑制剂（calpeptin）减弱。使用干涉反射/绿色荧光蛋白肌动蛋白显微镜评估剪切应力诱导的粘着斑 (FA) 和细胞骨架动力学后，我们发现钙蛋白酶或 PI3K 抑制通过稳定 FA 结构而损害剪切应力诱导的 FA 极化。此外，HUVEC 排列和细胞骨架重塑（伴随着钙蛋白酶介导的纽蛋白和踝蛋白裂解）也会因长时间施加剪切而引发，并因 m-钙蛋白酶敲低而受损。因此，这些结果表明，生理剪切应力会引起 HUVEC 中 m-钙蛋白酶的 Ca2+ 流入敏感激活。该活性主要通过 PI3K 途径促进；此外，它对于随后的 FA 重组和剪切条件下的细胞排列至关重要。
Proteolytic activity in sheared human umbilical vein endothelial cells (HUVECs) was measured using a fluorogenic substrate and laser scanning confocal microscopy to clarify the key role of an intracellular Ca2+- sensitive protease, calpain, in these cells in response to shear stress. Within physiological shear range, activity in the cells was enhanced in shear-dependent fashion. Short interfering RNA-induced silencing of m-calpain, but not of mu-calpain, suppressed the activity. Either removal of extracellular Ca2+ or application of an intracellular Ca2+ chelator (BAPTA/AM) or nonselective cation channel blocker (Gd3+) reduced proteolytic activity. Furthermore, activity was suppressed by phosphatidylinositol bisphosphate (PIP2) chelator (neomycin) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002); in contrast, activity, which was partially inhibited by ERK kinase inhibitor (U0126, PD98059), was unaffected by PLC inhibitor (U73122). Moreover, Akt phosphorylation downstream of PI3K, which was elicited by shear, was attenuated by neomycin but not by calpain inhibitor (calpeptin). Following assessment of shear stress-induced focal adhesion (FA) and cytoskeletal dynamics using interference reflection/green fluorescence protein-actin microscopy, we found that either calpain or PI3K inhibition impaired shear stress-induced polarization of FAs via stabilization of FA structures. Additionally, HUVEC alignment and cytoskeletal remodeling, which was accompanied by calpain-mediated cleavage of vinculin and talin, were also elicited by prolonged application of shear and impaired by m-calpain knockdown. Thus, these results revealed that physiological shear stress elicits Ca2+ influx-sensitive activation of m-calpain in HUVECs. This activity is facilitated primarily through the PI3K pathway; furthermore, it is essential for subsequent FA reorganization and cell alignment under shear conditions.