Rapamycin Regulates Endothelial Cell Migration through Regulation of the Cyclin-dependent Kinase Inhibitor p27Kip1
Rapamycin Regulates Endothelial Cell Migration through Regulation of the Cyclin-dependent Kinase Inhibitor p27Kip1
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DOI：
10.1074/jbc.m109.066621
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发表时间：
2010-04-16
影响因子：
4.8
通讯作者：
Woods, T. Cooper
中科院分区：
文献类型：
--
作者：
Moss, Stephanie C.;Lightell, Daniel J., Jr.;Woods, T. Cooper
雷帕霉素是一种抑制血管平滑肌细胞增殖和迁移的大环内酯类抗生素，临床上用于药物洗脱支架以抑制支架内再狭窄。尽管细胞迁移的抑制是预防再狭窄的一个优点，但它也会导致支架内皮化受损，这是目前药物洗脱支架技术的一个重大限制，需要延长抗血小板治疗。我们测量了雷帕霉素抑制人脐静脉内皮细胞（HUVECs）和人冠状动脉内皮细胞（HCAEC）向化学引诱物血管内皮细胞生长因子迁移的能力。虽然雷帕霉素急性给药没有影响，但暴露24小时可抑制HUVEC和HCAEC迁移。通过小干扰RNA破坏mTORC2也有效抑制HCAEC迁移。用雷帕霉素处理HCAEC这段时间，通过减少Thr磷酸化降解蛋白的靶向作用，导致细胞周期蛋白依赖性激酶抑制剂p27（Kip）增加（187）。与野生型对照相比，从表达p27（Kip1）的敲入小鼠中分离的EC表现出减少的迁移，其中该残基突变为丙氨酸，阻断了这种磷酸化。用小干扰RNA沉默p27（Kip1）阻断了雷帕霉素对迁移和管形成以及RhoA激活和细胞骨架重组的影响。我们的结论是，长期暴露的内皮细胞雷帕霉素增加p27（Kip1），反过来抑制RhoA激活，阻断细胞迁移和分化。这些数据阐明了雷帕霉素调节p27（Kip1）蛋白和细胞迁移的分子机制。
Rapamycin is a macrolide antibiotic that inhibits vascular smooth muscle cell proliferation and migration and that is used clinically on drug-eluting stents to inhibit in-stent restenosis. Although inhibition of cell migration is an asset in preventing restenosis, it also leads to impaired stent endothelialization, a significant limitation of current drug-eluting stent technology that necessitates prolonged antiplatelet therapy. We measured the ability of rapamycin to inhibit the migration of human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAEC) toward the chemoattractant vascular endothelial cell growth factor. Although acute administration of rapamycin had no effect, exposure for 24 h inhibited HUVEC and HCAEC migration. Disruption of the mTORC2 via small interfering RNA was also effective in inhibiting HCAEC migration. Treatment of HCAECs for this period with rapamycin produced an increase in the cyclin-dependent kinase inhibitor p27(Kip), through a decrease in the targeting of the protein for degradation by phosphorylation at Thr(187). ECs isolated from a knock-in mouse expressing p27(Kip1) with a mutation of this residue to an alanine, blocking this phosphorylation, exhibited reduced migration compared with wild-type controls. Silencing of p27(Kip1) with small interfering RNA blocked the effects of rapamycin on migration and tube formation as well as RhoA activation and cytoskeletal reorganization. We conclude that prolonged exposure of ECs to rapamycin increases p27(Kip1) and in turn inhibits RhoA activation, blocking cell migration and differentiation. These data elucidate the molecular mechanism underlying regulation of p27(Kip1) protein and cell migration by rapamycin.