Fatty acid-binding protein 4, a point of convergence for angiogenic and metabolic signaling pathways in endothelial cells

Abstract

Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment.

Keywords: Angiogenesis; DLL4; Endothelial Cell; FOXO; Fatty Acid-binding Protein; Metabolism; Notch Pathway; Vascular Endothelial Growth Factor (VEGF).

FIGURE 1.

DLL4-NOTCH signaling is a regulator of FABP4 mRNA and protein expression. HUVECs were grown on BSA- or DLL4-coated (BSAc or DLL4c) dishes for the indicated time points. Cells were harvested and analyzed for mRNA expression of FABP4 (A) or protein expression of DLL4 and FABP4 (B), and band densitometry analysis of FABP4 expression relative to the loading control in three independent experiments was performed (C). Values are expressed as mean ± S.D., *, p < 0.05; **, p < 0.01; ***, p < 0.001 (A). β-Actin was used as a loading control (B).

FIGURE 2.

DLL4-NOTCH-induced FABP4 is down-regulated by NOTCH signaling inhibition and by oxygen deprivation. HUVECs were grown on BSA- or DLL4-coated (BSAc or DLL4c) dishes in 21% oxygen, 1% oxygen, or 0.1% oxygen. Cells were treated with DBZ to inhibit NOTCH cleavage or vehicle control (DMSO). Cells were harvested and analyzed for protein expression of DLL4 and FABP4 (A), and band densitometry analysis of FABP4 protein expression relative to the loading control of three independent experiments was performed (B). β-Actin was used as a loading control. Values are expressed as mean ± S.D., *, p < 0.05; **, p < 0.01; ***, p < 0.001 (B).

FIGURE 3.

Inhibition of DLL4-NOTCH binding by a DLL4 blocking antibody reduces FABP4 induction by VEGFA. HUVECs were growth factor-starved, stimulated with BSA or VEGFA (50 ng/ml), and treated with DLL4 blocking antibody (anti-DLL4) or IgG control. Cells were harvested and analyzed for mRNA expression of DLL4 and FABP4. Values are expressed as mean ± S.D., *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

VEGFA-induced FABP4 is down-regulated by γ-secretase inhibition. HUVECs were growth factor-starved, stimulated with BSA or VEGFA (50 ng/ml), and treated with DBZ (10 nm) or vehicle control (DMSO). Cells were harvested and analyzed for mRNA expression of FABP4 (A), DLL4, HEY1, and PPARγ (C), or protein expression of DLL4 and FABP4 (B). β-Actin was used as a loading control (B). Values are expressed as mean ± S.D., *, p < 0.05 (A and C).

FIGURE 5.

Inhibition of ADAM10 and ADAM17 reduces FABP4 induction by VEGFA. HUVECs were growth factor-starved, stimulated with BSA or VEGFA (50 ng/ml), and treated with DBZ (A and B), INCB4298 (10 μm, targets ADAM17) (C), INCB3619 (5 μm, targets ADAM17/10) (C), or vehicle control (DMSO) as indicated. Cells were harvested and analyzed for protein expression of ADAM10 and ADAM17 (A, premature form = pADAM and mature form = mADAM), band intensity (three independent experiments (B)), or analyzed for mRNA expression of DLL4 and FABP4 (C). β-Actin was used as a loading control (A). Values are expressed as mean ± S.D., *, p < 0.05; **, p < 0.01 (C).

FIGURE 6.

AKT inhibition increases FABP4 expression. HUVECs were grown on BSA- or DLL4-coated (BSAc or DLL4c) dishes and treated with AKT inhibitor X (AKTiX) or vehicle control (DMSO). Cells were harvested and analyzed for protein expression of DLL4, AKT, and FABP4 and for the serine 473 phosphorylation status of AKT (pAKT). β-Actin was used as a loading control (A), and band densitometry analysis of three independent experiments was performed (B). Statistical analysis was performed by Friedman test for nonparametric data (pAKT p < 0.05; FABP4 p < 0.01).

FIGURE 7.

Regulation of FABP4 is dependent on FOXO1. HUVECs were growth factor-starved and stimulated with BSA or VEGFA (50 ng/ml) (A) or grown on BSA- or DLL4-coated (BSAc or DLL4c) (B and C) and were transfected with siRNA targeting FOXO1 (siFOXO1) or scrambled siRNA (control = ctr). Cells were harvested after 48 h and analyzed for mRNA expression of FOXO1 and FABP4 (A and B) and protein expression of DLL4, FOXO1 and FABP4 (C). β-Actin was used as a loading control (C). Values are expressed as mean ± S.D., *, p < 0.05; ***, p < 0.001 (A and B).

FIGURE 8.

NICD binds to specific FABP4 promoter regions adjacent to the FOXO1-binding motif. Location of RBPJκ-binding motifs (underlined, R-F-1, R-2, R-3A, R-3B, and R-3C) or adjacent RBPJκ- and FOXO1-binding motifs (R-F-1, FOXO1-binding motif in red) in the FABP4 promoter sequence are shown (A). HUVECs were grown on BSA- or DLL4-coated (BSAc or DLL4c) dishes for 16 h and were treated with DBA or vehicle control (DMSO). Cells were fixed and analyzed for NICD binding to the predicted binding sites R-F-1 and R2, known binding sites in DLL4 and HEY1 promoters, and a region in UBC promoter by chromatin immunoprecipitation (ChIP) (B). Values are expressed relative to percent input of the control condition (BSAc and DMSO) and as mean ± S.D., *, p < 0.05; **, p < 0.01 (B). Statistical analysis was performed by Friedman test for nonparametric data, and Dunn's post hoc test.

FIGURE 9.

Regulation and mechanism of FABP4 induction in endothelial cells is dependent on DLL4-NOTCH signaling and FOXO1. VEGFA induces DLL4 expression in endothelial cells, thereby promoting the paracrine activation of NOTCH signaling in adjacent cells and inducing indirect binding of NICD via a transcription factor complex to promoter regions of the FABP4 gene, to induce FABP4 transcription. Under basal levels of AKT activity, FOXO1 is present in the nucleus and promotes DLL4-NOTCH-dependent FABP4 transcription. AKT activation induces FOXO1 phosphorylation and cytoplasmic translocation and diminishes FABP4 transcription. AKT inhibition leads to FOXO1 being trapped in the nucleus and further promoting FABP4 transcription.