Cardiovascular Research Institute, Department of Cardiology, Shenyang Northern Hospital, Shenyang, China.
Exp Cell Res. 2009 Nov 15;315(19):3301-11. doi: 10.1016/j.yexcr.2009.09.013. Epub 2009 Sep 19.
We previously determined that the cellular repressor of E1A-stimulated genes, (CREG) plays a role in the maintenance of the mature phenotype of vascular smooth muscle cells (SMCs). This study aimed to identify the role of CREG in modulating the migration of SMCs. Recombinant virus-mediated CREG expression inhibited the cellular migration of cultured SMCs associated with down-regulated activity of matrix metalloproteinase-9 (MMP-9). In contrast, CREG knockdown via the retroviral transfer of short hairpin RNAs promoted cellular migration. Enzyme-linked immunosorbent assay and endocytosis analysis revealed that CREG knockdown attenuated the internalization and increased secretion of insulin-like growth factor (IGF)-II. Western blot analysis demonstrated that both phosphoinositide 3-kinase (PI3K) and phosphatase Akt were enhanced in CREG knockdown SMCs. Furthermore, the effect of CREG knockdown on SMC migration was abrogated in a dose-dependent manner by the addition of either IGF-II neutralizing antibody or the PI3K inhibitor, LY294002. These results indicate that the CREG knockdown-mediated increase in IGF-II secretion promoted cellular migration in SMCs via the PI3K/Akt signal pathway. Additionally, blockage of IGF-II binding to the mannose-6-phosphate/IGF-II receptor (M6P/IGF2R) by IGF2R antibody or recombinant IGF2R fragment attenuated the endocytosis of IGF-II in cells overexpressing CREG. This indicates that M6P/IGF2R is involved in the regulation of CREG-mediated IGF-II endocytosis. In summary, these data demonstrate for the first time that CREG plays a critical role in the inhibition of SMC migration, as well as maintaining SMCs in a mature phenotype. These results may provide a new therapeutic target for vascular disease associated with neointimal hyperplasia.
我们之前已经确定，E1A 刺激基因的细胞抑制剂（CREG）在维持血管平滑肌细胞（SMC）的成熟表型中发挥作用。本研究旨在确定 CREG 在调节 SMC 迁移中的作用。重组病毒介导的 CREG 表达抑制了与基质金属蛋白酶-9（MMP-9）活性下调相关的培养 SMC 的细胞迁移。相比之下，通过逆转录病毒转导短发夹 RNA 下调 CREG 表达促进了细胞迁移。酶联免疫吸附试验和内吞分析显示，CREG 下调减弱了胰岛素样生长因子（IGF）-II 的内化和增加了其分泌。Western blot 分析表明，在 CREG 下调的 SMC 中，磷酸肌醇 3-激酶（PI3K）和磷酸酶 Akt 均增强。此外，通过添加 IGF-II 中和抗体或 PI3K 抑制剂 LY294002，以剂量依赖的方式消除了 CREG 下调对 SMC 迁移的影响。这些结果表明，通过 PI3K/Akt 信号通路，CREG 下调介导的 IGF-II 分泌增加促进了 SMC 的细胞迁移。此外，IGF2R 抗体或重组 IGF2R 片段阻断 IGF-II 与甘露糖-6-磷酸/IGF-II 受体（M6P/IGF2R）的结合，减弱了 CREG 过表达细胞中 IGF-II 的内吞作用。这表明 M6P/IGF2R 参与了 CREG 介导的 IGF-II 内吞作用的调节。总之，这些数据首次表明 CREG 在抑制 SMC 迁移以及维持 SMC 成熟表型方面发挥着关键作用。这些结果可能为与血管内膜增生相关的血管疾病提供新的治疗靶点。
