FoxO3 modulates endothelial gene expression and function by direct and indirect mechanisms . Homo sapiens

FoxO transcription factors represent targets of the PI3K/PKB survival pathway controlling important biological processes such as stress responses, cell cycle progression, apoptosis, vascular remodelling and metabolism. Recent studies have demonstrated the existence of alternative mechanisms of FoxO-dependent gene expression beyond classical binding to a FoxO-responsive DNA binding element (FRE). Here we explored the relative contribution of those mechanisms by comparing the transcriptional responses to conditional activation of FoxO3 and a corresponding FRE-binding mutant in primary human endothelial cells. Microarray analysis revealed several functional gene clusters regulated in absence of an intact DNA-binding domain. Notably, both mutants triggered apoptosis albeit with different efficiencies. This was associated with regulation of overlapping and distinct proapototic gene clusters. Subsequent analysis demonstrated important roles for the Bcl2-like family members BIM and NOXA in this process. Remarkably, BIM was effectively induced by the FoxO3 FRE-binding mutant and BIM depletion could rescue its proapoptotic effect. Our study provides the first comprehensive analysis of alternatively regulated FoxO3 targets in primary cells and underscores the importance of such genes for endothelial function and integrity. Overall design: HUVEC were infected in 4 independent experiments with either empty pBabe puro vector, pBP-FoxO3.A3.ER, or pBP FoxO3.A3.ER.H212R in three consecutive rounds and 72h post infection.

FoxO转录因子（FoxO transcription factors）是PI3K/PKB存活通路的靶标，该通路调控应激反应、细胞周期进程、细胞凋亡、血管重构及代谢等重要生物学过程。近期研究证实，除经典结合FoxO应答DNA结合元件（FRE）之外，还存在FoxO依赖型基因表达的非经典调控机制。本研究通过比较原代人内皮细胞中FoxO3的条件性激活与对应FRE结合突变体的转录应答，探究了此类调控机制的相对贡献。微阵列分析显示，在缺失完整DNA结合结构域的情况下，存在多个受调控的功能基因簇。值得注意的是，两种突变体均能诱导细胞凋亡，但诱导效率存在差异，这与重叠及独特的促凋亡基因簇的调控密切相关。后续分析证实，Bcl2家族同源成员BIM与NOXA在此过程中发挥重要作用。尤为关键的是，FoxO3的FRE结合突变体可有效诱导BIM表达，而BIM的敲低能够挽救其促凋亡效应。本研究首次在原代细胞中对受非经典调控的FoxO3靶基因进行了全面分析，并强调了此类基因在内皮细胞功能与完整性维持中的重要性。整体实验设计：于4次独立实验中，将人脐静脉内皮细胞（HUVEC）分别以空载pBabe嘌呤霉素载体、pBP-FoxO3.A3.ER或pBP FoxO3.A3.ER.H212R进行三轮连续感染，并于感染后72小时开展后续检测。