Fosbrink Matthew, Niculescu Florin, Rus Violeta, Shin Moon L, Rus Horea
Department of Neurology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
J Biol Chem. 2006 Jul 14;281(28):19009-18. doi: 10.1074/jbc.M602055200. Epub 2006 May 2.
Migration and proliferation of aortic endothelial cells (AEC) are critical processes involved in angiogenesis, atherosclerosis, and postangioplasty restenosis. Activation of complement and assembly of the C5b-9 complement complex have been implicated in the pre-lesional stage of atherogenesis and progression of the atherosclerotic lesion. We have shown that C5b-9 induces proliferation and activates phosphatidylinositol 3-kinase (PI3K), but it is unknown whether this can lead to activation of Akt in AEC, a major downstream target of PI3K, or if C5b-9 can induce the migration of AEC, a critical step in angiogenesis. In this study, we show that C5b-9 induces AEC proliferation and migration and also activates the PI3K/Akt pathway. C5b-9 activates Akt as shown by in vitro kinase assay and phosphorylation of Ser-473. C5b-9-induced cell cycle activation was inhibited by pretreatment with LY294002 (PI3K inhibitor), SH-5 (Akt inhibitor), or transfection with Akt siRNA. These data suggests that the PI3K/Akt pathway is required for C5b-9-induced cell cycle activation. FOXO1, a member of forkhead transcription factor family, was phosphorylated at Ser-256 and inactivated after C5b-9 stimulation as shown by a decrease in DNA binding and cytoplasmic relocalization. Cytoplasmic relocalization was significantly reduced after pretreatment with LY294002, SH-5, or transfection with Akt siRNA. Silencing FOXO1 expression using siRNA stimulated AEC proliferation and regulated angiogenic factor release. Our data indicate that C5b-9 regulation of the cell cycle activation in AEC through Akt pathway is dependent on inactivation of FOXO1.
主动脉内皮细胞（AEC）的迁移和增殖是血管生成、动脉粥样硬化和血管成形术后再狭窄所涉及的关键过程。补体的激活和C5b-9补体复合物的组装与动脉粥样硬化形成的病变前期阶段以及动脉粥样硬化病变的进展有关。我们已经表明C5b-9可诱导增殖并激活磷脂酰肌醇3激酶（PI3K），但尚不清楚这是否会导致PI3K的主要下游靶点AEC中的Akt激活，或者C5b-9是否能诱导AEC迁移，这是血管生成中的关键步骤。在本研究中，我们表明C5b-9可诱导AEC增殖和迁移，还可激活PI3K/Akt信号通路。如体外激酶测定和Ser-473磷酸化所示，C5b-9可激活Akt。用LY294002（PI3K抑制剂）、SH-5（Akt抑制剂）预处理或用Akt siRNA转染可抑制C5b-9诱导的细胞周期激活。这些数据表明PI3K/Akt信号通路是C5b-9诱导的细胞周期激活所必需的。叉头转录因子家族成员FOXO1在Ser-256处被磷酸化，并在C5b-9刺激后失活，这表现为DNA结合减少和细胞质重新定位。用LY294002、SH-5预处理或用Akt siRNA转染后，细胞质重新定位显著减少。使用siRNA沉默FOXO1表达可刺激AEC增殖并调节血管生成因子的释放。我们的数据表明，C5b-9通过Akt信号通路对AEC细胞周期激活的调节依赖于FOXO1的失活。
