Role of CPI-17 in the regulation of endothelial cytoskeleton
Role of CPI-17 in the regulation of endothelial cytoskeleton
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DOI：
10.1152/ajplung.00398.2003
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发表时间：
2004-11-01
影响因子：
4.9
通讯作者：
Verin, AD
中科院分区：
文献类型：
--
作者：
Kolosova, IA;Ma, SF;Verin, AD
我们先前已经表明，肌球蛋白轻链（MLC）磷酸酶（MLCP）在激动剂介导的内皮渗透性和细胞骨架组织的调节中起关键作用（贝林AD，Patterson CE，Day MA和Garcia JG. Am J Physiol Lung Cell Mol Physiol 269：L99-L108，1995）。然而，内皮MLCP调节的分子机制还不完全清楚。在本研究中，我们发现，类似于平滑肌，肺微血管内皮细胞表达特异性内源性MLCP抑制剂CPI-17。为了阐明CPI-17在内皮细胞骨架调节中的作用，将全长CPI-17质粒瞬时转染到内源性蛋白背景较低的肺动脉内皮细胞中。CPI-17在非刺激条件下对细胞骨架无影响。然而，与未转染细胞相比，直接PKC激活剂PMA刺激转染细胞引起F-肌动蛋白应力纤维、粘着斑和MLC磷酸化的显著增加。炎症激动剂组胺和凝血酶（在较小程度上）能够激活CPI-17。组胺引起的CPI-17磷酸化比凝血酶强。抑制分析表明，PKC更显着地有助于激动剂诱导的CPI-17磷酸化比Rho激酶。显性负性PKC-α消除了CPI-17对肌动蛋白细胞骨架的作用，表明PKC-α亚型最有可能是CPI-17在内皮中活化的原因。肺微血管内皮细胞中内源性CPI-17的消耗显著减弱组胺诱导的内皮通透性增加。总之，这些数据表明，PKC/CPI-17介导的途径在组胺触发的细胞骨架重排导致肺微血管屏障受损的潜在重要性。
We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin AD, Patterson CE, Day MA, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 269: L99-L108, 1995). The molecular mechanisms of endothelial MLCP regulation, however, are not completely understood. In this study we found that, similar to smooth muscle, lung microvascular endothelial cells expressed specific endogenous inhibitor of MLCP, CPI-17. To elucidate the role of CPI-17 in the regulation of endothelial cytoskeleton, full-length CPI-17 plasmid was transiently transfected into pulmonary artery endothelial cells, where the background of endogenous protein is low. CPI-17 had no effect on cytoskeleton under nonstimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions, and MLC phosphorylation compared with untransfected cells. Inflammatory agonist histamine and, to a much lesser extent, thrombin were capable of activating CPI-17. Histamine caused stronger CPI-17 phosphorylation than thrombin. Inhibitory analysis revealed that PKC more significantly contributes to agonist-induced CPI-17 phosphorylation than Rho-kinase. Dominant-negative PKC-alpha abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC-alpha isoform is most likely responsible for CPI-17 activation in the endothelium. Depletion of endogenous CPI-17 in lung microvascular endothelial cell significantly attenuated histamine-induced increase in endothelial permeability. Together these data suggest the potential importance of PKC/CPI-17-mediated pathway in histamine-triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise.