FOXO3 Modulates Endothelial Gene Expression and Function by Classical and Alternative Mechanisms

Abstract

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.The FOXOs represent an evolutionarily conserved subfamily of forkhead transcription factors unified by the presence of a highly homologous DNA-binding domain termed the "forkhead box." In contrast to invertebrates, which only express a single FOXO gene (dfoxo in Drosophila melanogaster and daf-16 in Caenorhabditis elegans) mammals contain four distinct isoforms: FOXO1, FOXO3, FOXO4 and the atypical FOXO6, which shows divergent modes of regulation and expression (1). FOXOs are regulated by various posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. Among those, phosphorylation by the insulin receptor/phosphatidylinositol 3-kinase/protein kinase B cascade provides a master switch of FOXO activity. Growth factors or insulin stimulation leads to activation of this pathway and triggers protein kinase B-dependent FOXO phosphorylation at three distinct residues, followed by nuclear exclusion and proteasomal degradation (2). In contrast, the absence of growth factors or treatment with inhibitors of the phosphatidylinositol 3-kinase/protein kinase B pathway results in silencing...

Pro-apoptotic BIM is an essential initiator of physiological endothelial cell death independent of regulation by FOXO3

“…Despite in vitro evidence that FOXO3 is sufficient to induce Bim-dependent apoptosis in endothelial cells, 27,29 we found no evidence that FOXO3 was required for BIM-dependent endothelial cell apoptosis in vivo. It has been reported that FOXO3 can induce BIM expression in endothelial cells independently of binding to the FOXO consensus sequence.…”

“…It has been reported that FOXO3 can induce BIM expression in endothelial cells independently of binding to the FOXO consensus sequence. 29 Although we can exclude an indirect role for FOXO3 in BIM-dependent endothelial apoptosis, we cannot exclude such a role for FOXO1 or FOXO4. The importance of FOXO1 and FOXO4 in Bim-dependent endothelial death is questionable, however.…”

“…26,27 While endothelial cells express multiple FOXO family members, [26][27][28] in vitro studies implicate FOXO3 as the predominant regulator of Bim in this cell type: knockdown of Foxo3 but not Foxo1 resulted in reduced Bim expression in endothelial cells, 27 whereas overexpression of constitutively active FOXO3 induced Bim expression. 29 The requirement for FOXO3 in endothelial cell death in a physiologically relevant context, however, remains to be proven.…”

FOXO3 as a new IKK‐ε‐controlled check‐point of regulation of IFN‐β expression

“…In response to various stimuli, including TLR induction by microbial and viral pathogen, DCs produce proinflammatory cytokines and type I IFNs [30]. FOXO3 was previously reported to participate in the regulation of proinflammatory cytokine production in DCs and endothelial cells [10,29,31]. Here, we discover that FOXO3 also has the ability to inhibit IFN-β production in human MDDCs.…”

“…7B). Of interest, most of the genes involved in proliferation and the cell-cycle regulation that are downregulated by FOXO3, are not dependent on FOXO3 interactions with DNA but rather on its protein-protein interaction [31] with transcription factors like p53 and β-catenin [34]. This suggests that DNA-binding independent mode of FOXO3 function may be a common mechanism for FOXO3-mediated transcriptional repression.…”