Department of Dermatology, University Medical Center Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany.
J Biol Chem. 2010 Apr 2;285(14):10163-78. doi: 10.1074/jbc.M109.056663. Epub 2010 Feb 1.
FOXO transcription factors represent targets of the phosphatidylinositol 3-kinase/protein kinase B survival pathway controlling important biological processes, such as cell cycle progression, apoptosis, vascular remodeling, stress responses, and metabolism. Recent studies suggested the existence of alternative mechanisms of FOXO-dependent gene expression beyond classical binding to a FOXO-responsive DNA-binding element (FRE). Here we analyzed the relative contribution of those mechanisms to vascular function by comparing the transcriptional and cellular responses to conditional activation of FOXO3 and a corresponding FRE-binding mutant in human primary endothelial cells. We demonstrate that FOXO3 controls expression of vascular remodeling genes in an FRE-dependent manner. In contrast, FOXO3-induced cell cycle arrest and apoptosis occurs independently of FRE binding, albeit FRE-dependent gene expression augments the proapoptotic response. These findings are supported by bioinformatical analysis, which revealed a statistical overrepresentation of cell cycle regulators and apoptosis-related genes in the group of co-regulated genes. Molecular analysis of FOXO3-induced endothelial apoptosis excluded modulators of the extrinsic death receptor pathway and demonstrated important roles for the BCL-2 family members BIM and NOXA in this process. Although NOXA essentially contributed to FRE-dependent apoptosis, BIM was effectively induced in the absence of FRE-binding, and small interfering RNA-mediated BIM depletion could rescue apoptosis induced by both FOXO3 mutants. These data suggest BIM as a critical cell type-specific mediator of FOXO3-induced endothelial apoptosis, whereas NOXA functions as an amplifying factor. Our study provides the first comprehensive analysis of alternatively regulated FOXO3 targets in relevant primary cells and underscores the importance of such genes for endothelial function and integrity.
叉头框转录因子（FOXO transcription factors）是磷酸肌醇 3-激酶/蛋白激酶 B 生存途径的靶标，该途径控制着细胞周期进程、细胞凋亡、血管重塑、应激反应和代谢等重要的生物学过程。最近的研究表明，除了与 FOXO 反应性 DNA 结合元件（FOXO-responsive DNA-binding element，FRE）的经典结合之外，FOXO 依赖性基因表达存在替代机制。在这里，我们通过比较条件性激活 FOXO3 和相应的 FRE 结合突变体在人原代内皮细胞中的转录和细胞反应，分析了这些机制对血管功能的相对贡献。我们证明，FOXO3 以依赖 FRE 的方式控制血管重塑基因的表达。相比之下，FOXO3 诱导的细胞周期停滞和细胞凋亡独立于 FRE 结合，但 FRE 依赖性基因表达增强了促凋亡反应。这些发现得到了生物信息学分析的支持，该分析揭示了细胞周期调节剂和凋亡相关基因在共同调控基因组中存在统计学上的过度表达。对 FOXO3 诱导的内皮细胞凋亡的分子分析排除了细胞外死亡受体途径的调节剂，并证明了 BCL-2 家族成员 BIM 和 NOXA 在该过程中的重要作用。虽然 NOXA 主要有助于 FRE 依赖性凋亡，但在没有 FRE 结合的情况下，BIM 被有效诱导，并且小干扰 RNA 介导的 BIM 耗竭可以挽救由两种 FOXO3 突变体诱导的凋亡。这些数据表明 BIM 是 FOXO3 诱导的内皮细胞凋亡的关键细胞类型特异性介质，而 NOXA 则作为放大因子。我们的研究首次对相关原代细胞中替代调节的 FOXO3 靶标进行了全面分析，并强调了这些基因对内皮功能和完整性的重要性。
