FoxO3 modulates endothelial gene expression and function by direct and indirect mechanisms

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. 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.