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.
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"It is therefore likely that the FOXO-interference activity of PAX3-FOXO1 is due to the FOXO1 moiety of PAX3-FOXO1. Non-classical gene-regulation exerted by a non-DNA-binding FOXO mutant has been described [46,47,48]. These surprising effects were shown on genes without FOXO DNA-binding elements (IRS) in their promoter and were explained by FOXO's capacity (and also mutant FOXO lacking DNA-binding capacity) to bind gene promoters indirectly by making complexes with other DNA-binding proteins targeted to these promoters. "
[Show abstract][Hide abstract]ABSTRACT: PAX3-FOXO1 (PAX3-FKHR) is the fusion protein produced by the genomic translocation that characterizes the alveolar subtype of Rhabdomyosarcoma, a pediatric sarcoma with myogenic phenotype. PAX3-FOXO1 is an aberrant but functional transcription factor. It retains PAX3-DNA-binding activity and functionally overlaps PAX3 function while also disturbing it, in particular its role in myogenic differentiation. We herein show that PAX3-FOXO1 interferes with normal FOXO function. PAX3-FOXO1 affects FOXO-family member trans-activation capability and the FOXO-dependent TGF-β response. PAX3-FOXO1 may contribute to tumor formation by inhibiting the tumor suppressor activities which are characteristic of both FOXO family members and TGF-β pathways. The recognition of this mechanism raises new questions about how FOXO family members function.
"In addition, up-regulation of SMAD2 and IGFBP-3 by PAX3 knock-down could mediate proliferation inhibition and apoptosis via mediating TGF-b, p53 and BCL2 family-dependent apoptosis [28, 29]. Up-regulation of FOXO3 by PAX3 knock-down could also induce BIM expression and lead to apoptosis . Taken together, PAX3 silencing activates multiple apoptotic signalling pathways, while simultaneously inhibiting anti-apoptotic signalling pathways, thereby inducing apoptosis in neuroblastoma cells. "
[Show abstract][Hide abstract]ABSTRACT: Transcription factor PAX3/Pax3 contributes to diverse cell lineages during embryonic development and is important in tumourigenesis. We found that PAX3 is re-expressed in neuroblastoma and malignant neuroblastic (N-type) neuroblastoma cells had significantly higher PAX3 protein expression than their benign substrate-adherent (S-type) counterparts. Knock-down of PAX3 expression by siRNA transfection resulted in persistent cell growth inhibition in both types of neuroblastoma cell, owing to G1 cell cycle arrest and progressive apoptosis. Inhibition of PAX3 expression significantly decreased the attachment of S-type SH-EP1 cells to extra-cellular matrix proteins, fibronectin, laminin and collagen IV. Migration and invasion of both neuroblastoma cell types were markedly reduced after PAX3 down-regulation. PAX3 knock-down significantly augmented the cytotoxic effect of chemotherapeutic agents, etoposide, vincristine and cisplatin, commonly used to treat neuroblastoma. Microarray analyses revealed that particularly signalling pathways involving cell cycle, apoptosis, cell adhesion, cytoskeletal remodelling and development were altered by PAX3 down-regulation. Changes in PAX3 downstream genes identified by microarray analyses were validated in 47 genes by quantitative PCR. These novel findings lead us to propose that PAX3 might contribute to oncogenic characteristics of neuroblastoma cells by regulating a variety of crucial signalling pathways.
Full-text · Article · Nov 2013 · Journal of Cellular and Molecular Medicine
"These findings indicate that, at early or mild stages of stress, FoxO3a is required to initiate a proliferative cellular program, but that, upon onset of severe or prolonged stress, FoxO3a may contribute to a proapoptotic program shift. Interestingly, a recent report suggested that direct binding of FoxO3a to FHRE may enhance transcriptional activation of genes involved in vascular remodeling in endothelial cells, whereas the proapoptotic functions of FoxO3a appeared to be mediated independently of FHRE binding (Czymai et al., 2010). Our findings that FoxO3a selectively increases MMP2 expression via binding to an FIGURE 7: FoxO3a is required for vascular outgrowth. "
[Show abstract][Hide abstract]ABSTRACT: The vasoactive peptide urotensin-II (U-II) has been associated with vascular remodeling in different cardiovascular disorders. Although U-II can induce reactive oxygen species (ROS) by the NADPH oxidase NOX4 and stimulate smooth muscle cell (SMC) proliferation, the precise mechanisms linking U-II to vascular remodeling processes remain unclear. Forkhead Box O (FoxO) transcription factors have been associated with redox signaling and control of proliferation and apoptosis. We thus hypothesized that FoxOs are involved in the SMC response toward U-II and NOX4. We found that U-II and NOX4 stimulated FoxO activity and identified matrix metalloproteinase-2 (MMP2) as target gene of FoxO3a. FoxO3a activation by U-II was preceded by NOX4-dependent phosphorylation of c-Jun NH(2)-terminal kinase and 14-3-3 and decreased interaction of FoxO3a with its inhibitor 14-3-3, allowing MMP2 transcription. Functional studies in FoxO3a-depleted SMCs and in FoxO3a(-/-) mice showed that FoxO3a was important for basal and U-II-stimulated proliferation and vascular outgrowth, whereas treatment with an MMP2 inhibitor blocked these responses. Our study identified U-II and NOX4 as new activators of FoxO3a, and MMP2 as a novel target gene of FoxO3a, and showed that activation of FoxO3a by this pathway promotes vascular growth. FoxO3a may thus contribute to progression of cardiovascular diseases associated with vascular remodeling.
Full-text · Article · Sep 2011 · Molecular biology of the cell