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.

FIGURE 1.

Conditional FOXO3 activation induces apoptosis in primary ECs irrespective of direct FRE-binding. HUVECs were retrovirally infected with either empty vector, FOXO3.A3.ER or FOXO3.A3.ER.H212R, selected by puromycin, and reseeded. A, luciferase assay demonstrating the incapability of the H212R mutant to transactivate a forkhead-responsive 6×DBE-luc reporter upon 16-h treatment with 4OHT. Data represent -fold average luciferase activation ± S.D. of a 6× DBE-luc reporter transfected into the differently infected cells. Luciferase values were each normalized to activity of a co-transfected Renilla luciferase reporter and related to unstimulated empty vector-infected cells. B, HA tag-specific chromatin immunoprecipitation after 12 h of 4OHT treatment showing binding of HA-FOXO3.A3.ER but not HA-FOXO3.A3.ER.H212R to an established FOXO-responsive region (6) in the IGFBP1 promoter. Data are represented as -fold enrichment of binding related to 4OHT-treated vector controls. C, qRT-PCR demonstrating expression of IGFBP1, normalized to GAPDH expression after 12 h of 4OHT treatment. Data display average -fold regulation ± S.D. related to unstimulated vector. D, Western blot, showing expression of p27^Kip1 and cleavage of caspase-3 32 h after 4OHT treatment. E, cell cycle profiles, displaying the percentage of subdiploid DNA content assessed by flow cytometric analysis of PI-stained cells 48 h after 4OHT treatment. F, colony formation assay after 6 days (6d) of 4OHT treatment. All data are derived from at least three independent experiments. Statistical significance was calculated by Student's t test.

FIGURE 2.

Functional annotation clustering reveals the presence of classically and alternatively regulated functional gene clusters. HUVECs were retrovirally infected with either empty vector, FOXO3.A3.ER, or FOXO3.A3.ER.H212R and reseeded after puromycin selection. Total RNA from three independent experiments was isolated after 12 h of 4OHT treatment, and global gene expression was determined by oligonucleotide microarray analysis, as described under “Experimental Procedures.” Gene profiles were analyzed using DAVID as described under “Experimental Procedures.” The enrichment score on the x axis ranks the overrepresentation of a cluster composed of functionally similar GO groups in relation to the total number of genes on the chip. Functional annotation clusters were assigned by the subset GO group designation with the highest significant p value.

FIGURE 3.

FOXO3 induces genes involved in migration, vascular remodeling, and leukocyte differentiation. HUVECs were retrovirally infected with the indicated constructs and puromycin-selected prior to reseeding. A–C, qRT-PCR showing expression of genes involved in migration, vascular remodeling, and leukocyte differentiation by classical and alternative mechanisms after 12 h of 4OHT treatment. Gene expression was normalized to GAPDH expression, and data are presented as average -fold regulation ± S.D. as compared with unstimulated vector controls and are derived from at least three independent experiments. Statistical significances were calculated by Student's t test. n.s., not significant.

FIGURE 4.

Conditional activation of FOXO3. H212R.ER induces a G₁ cell cycle arrest in primary human ECs. HUVECs were retrovirally infected with either empty vector, FOXO3.A3.ER or FOXO3.A3.ER.H212R, selected by puromycin, and reseeded. A, flow cytometric quantification of BrdUrd positivity after stimulation with 4OHT for the indicated times. The percentage of BrdUrd-positive cells (i.e. cells undergoing replication) is indicated. B, statistical analysis of BrdUrd-positive cells after activation of FOXO3. Data represent mean values of BrdUrd positivity ± S.D. and are derived from three independent experiments. C, qRT-PCRs, showing repression of endothelial CCND1 and PCNA mRNA 12 h after 4OHT treatment. Gene expression was normalized to GAPDH expression and is presented as average -fold expression ± S.D. of three independent experiments in relation to unstimulated vector controls. Statistical significances were determined using Student's t test. n.s., not significant.

FIGURE 5.

The extrinsic death receptor pathway is not involved in FOXO3-induced apoptosis. HUVECs were retrovirally infected with the indicated constructs, puromycin-selected, and reseeded for the respective experiments. A, Western blot, showing cFLIP and caspase-8 levels after exposure of cells to medium or 4OHT for 16 or 32 h. Additionally, stimulations with TRAIL (100 ng/ml, 3 h) or TRAIL + NiCl₂ (Ni) (1.5 mm) are shown as positive control for cFLIP or caspase-8 cleavage, respectively. B, cell cycle profiles of HUVECs, infected as indicated and treated with 10 μm LY392002 for 48 h. The percentage of subdiploid cells was determined by flow cytometric analysis of PI-stained cells. C, Western blot showing expression of FOXO3, cFLIP_L, and cleaved caspase-3 after 32 h of 4OHT treatment. D, cell cycle profiles after 48 h of 4OHT treatment. The percentage of PI-stained cells with a subdiploid DNA content was assessed by flow cytometry. All Western blots and cell cycle profiles are representative of three independent experiments.

FIGURE 6.

The Bcl-2 family member BIM contributes to FOXO3-induced apoptosis. HUVECs were retrovirally transduced with either empty vector, FOXO3.A3.ER, or FOXO3.A3.ER.H212R and puromycin-selected prior to reseeding. A, luciferase assay, showing BIM promoter activity normalized to Renilla activity after co-transfection of BIM- and Renilla-luciferase reporter gene constructs and 4OHT treatment for 16 h. B, qRT-PCR demonstrating BIM expression, normalized to GAPDH expression after 12 h of 4OHT treatment. C, Western blot, indicating expression of BIM 32h after 4OHT treatment. D, HA tag-specific chromatin immunoprecipitation after 12 h of 4OHT treatment showing binding of HA-FOXO3.A3.ER and HA-FOXO3.A3.ER.H212R to a conserved region in the BIM promoter containing a reported FRE-related sequence (12). E, Western blot, showing knockdown levels of BIM protein and cleavage of caspase-3 protein in response to transfection with either siRNA against BIM or scrambled control siRNA and 4OHT treatment for 24 h. F, cell cycle profiles, displaying the percentage of cells with subdiploid DNA content after transfection of siRNA against BIM or scrambled siRNA followed by 48-h treatment with 4OHT. G, statistical analysis of apoptosis suppression upon knockdown of BIM. Relative levels of 4OHT-induced apoptosis were calculated in relation to subdiploidy values of the individual scrambled siRNA-transfected cells expressing the indicated FOXO mutants (arbitrarily set to 1). Statistical data in A and B are presented as mean -fold regulation ± S.D. in relation to unstimulated vector. In D, OHT-treated vector-infected cells served as reference. All data are derived from at least three independent experiments. Significances were calculated by Student's t test.

FIGURE 7.

The Bcl-2 family member NOXA contributes to FOXO3-induced apoptosis. HUVECs were retrovirally infected with the indicated constructs and reseeded after puromycin selection. A, qRT-PCR showing NOXA expression, normalized to GAPDH expression after 12 h of 4OHT treatment. B, qRT-PCR demonstrating knockdown of NOXA mRNA after transfection of either siRNA against NOXA or scrambled siRNA and 4OHT treatment for 12 h. Data in A and B are presented as average -fold regulation ± S.D. related to unstimulated vector controls and are derived from at least three independent experiments. C, Western blot, displaying cleavage of caspase-3 upon transfection with either siRNA against NOXA or scrambled siRNA, followed by 4OHT treatment for 24 h. D, cell cycle profiles, showing the amount of PI-stained cells with subdiploid DNA content as determined by flow cytometry of the differently infected and siRNA-transfected cells 48 h after treatment with 4OHT. E, statistical analysis of apoptosis suppression by NOXA siRNA. Relative apoptosis was calculated as described in the legend to Fig. 6G. Statistical significances were calculated using Student's t test.