Feed-forward signaling by membrane-bound ligand receptor circuit: the case of NOTCH DELTA-like 4 ligand in endothelial cells

Abstract

The DELTA like-4 ligand (DLL4) belongs to the highly conserved NOTCH family and is specifically expressed in the endothelium. DLL4 regulates crucial processes in vascular growth, including endothelial cell (EC) sprouting and arterial specification. Its expression is increased by VEGF-A. In the present study, we show that VEGF-induced DLL4 expression depends on NOTCH activation. VEGF-induced DLL4 expression was prevented by the blockage of NOTCH signaling with γ-secretase or ADAM inhibitors in human cardiac microvascular ECs. Similar to VEGF-A, recombinant DLL4 itself stimulated NOTCH signaling and resulted in up-regulation of DLL4, suggesting a positive feed-forward mechanism. These effects were abrogated by NOTCH inhibitors but not by inhibition of VEGF signaling. NOTCH activation alone suffices to induce DLL4 expression as illustrated by the positive effect of NOTCH intracellular domain (NICD)-1 or -4 overexpression. To discriminate between NICD/RBP-Jκ and FOXC2-regulated DLL4 expression, DLL4 promoter activity was assessed in promoter deletion experiments. NICD induced promoter activity was dependent on RBP-Jκ site but independent of the FOXC2 binding site. Accordingly, constitutively active FOXC2 did not affect DLL4 expression. The notion that the positive feed-forward mechanism might propagate NOTCH activation to neighboring ECs was supported by our observation that DLL4-eGFP-transfected ECs induced DLL4 expression in nontransfected cells in their vicinity. In summary, our data provide evidence for a mechanism by which VEGF or ligand-induced NOTCH signaling up-regulates DLL4 through a positive feed-forward mechanism. By this mechanism, DLL4 could propagate its own expression and enable synchronization of NOTCH expression and signaling between ECs.

FIGURE 1.

VEGF-A₁₆₅-induced DLL4 expression is VEGFR-2- and NOTCH signaling-mediated. A, DLL4 mRNA expression in HCMvECs treated with VEGF-A₁₆₅, VEGF-E or PlGF-2 compared with the expression of nontreated cells as control. B, DLL4 mRNA expression in HCMvECs treated with VEGF-A₁₆₅ or VEGF-A₁₆₅ along with IMC-1121b (hVEGFR-2 inhibitor), or IMC-18F1 (hVEGFR-1 inhibitor) or IMC-1121b and IMC-18F1. C, DLL4 mRNA expression in VEGFR-2/PAECs (right) and VEGFR-1/PAECs (left) upon stimulation with or without VEGF-A₁₆₅. D, DLL4 mRNA expression in HCMvECs stimulated with or without VEGF-A₁₆₅ and VEGF-A₁₆₅ along with L685,458 (γ-secretase inhibitor), GI254023X (ADAM-10 inhibitor) or GW280264X (GW; ADAM-10/ADAM-17 inhibitor). E, luciferase activity of HCMvECs cell lysates transiently transfected with RBP-Jκ -luciferase reporter construct and cultured with or without VEGF-A₁₆₅ or VEGF-A₁₆₅ along with L685,458 (γ-secretase inhibitor). F, HES-1 mRNA expression in HCMvECs stimulated with or without VEGF-A₁₆₅ and VEGF-A₁₆₅ along with L685,458 (γ-secretase inhibitor), GI254023X (ADAM-10 inhibitor), or GW280264X (ADAM-10/ADAM-17 inhibitor). Data represent mean ± SE (n = 3). One-way ANOVA test was used. *, significantly different from control p < 0.05; #, significantly different from VEGF-A₁₆₅; p < 0.05. Ctrl, control.

FIGURE 2.

NICD-1- and NICD-4-induced DLL4 expression in HMvECs. A and B, DLL4 mRNA expression in HCMvECs transiently transfected with NICD-1, NICD-4, or pcDNA empty vector as control with or without L685,458. Data represent mean ± SE (n = 3). One-way ANOVA test was used. *, significantly different from control (mock) p < 0.05. C, Western blot analysis of protein extracts from HCMvECs transfected with NICD-1 and NICD-4. Membranes were probed with anti-NOTCH-1 (upper left panel), anti-NICD-4 (upper right panel), or anti-DLL4 (lower panel).

FIGURE 3.

Induction of DLL4 promoter activity by NICD-1 and NICD-4. A, luciferase assay activity from PAE cells contransfected with a 6-kb DLL4 promoter luciferase reporter or shortened fragments of the 6-kb promoter containing three (−2616), two (−1587), one (−931), or none (−931-D) RBP-Jκ binding site along with NICD-1, NICD-4, or empty pcDNA (mock) vector. Schematic drawing of the 6-kb DLL4 promoter and of the shortened fragments −2616, −1587, −931, and −931-D (relative to the transcriptional initiation site) with potential RBP-Jκ (R) sites indicated by red bars, FBE is indicated by a green bar, and deletion of (R) is indicated by an X. B, DLL4 mRNA expression in PAECs transiently transfected with NICD-1 and NICD-4 and pcDNA as control. Data represent mean ± SE (n = 3). One-way ANOVA test was used. *, significantly different from control (mock), p < 0.05. C, Western blot analysis of protein extracts from PAECs transfected with NICD-1 (upper panel) and NICD-4 (lower panel). Results are from one representative experiment. D, DLL4 mRNA expression in HCMvECs (left panel) and PAECs (right panel) transiently transfected with constitutively active FOXC2 (caFOXC2) and empty vector as control.

FIGURE 4.

rDLL4 activated NOTCH signaling induced DLL4 expression. A, DLL4 mRNA expression in HCMvECs stimulated with BSA as control, rDLL4, or rDLL4 along with L685,458 (γ-secretase inhibitor), or GI254023X (ADAM-10 inhibitors) or GW280264X (ADAM-10 and ADAM-17). B, luciferase activity of HCMvEC cell lysates transiently transfected with RBP-Jκ-luciferase reporter construct and stimulated with BSA as control, rDLL4 protein or with rDLL4 and L685,458. C, DLL4 mRNA expression in HCMvECs stimulated with BSA as control, rDLL4 or rDLL4 along with IMC-1121b (hVEGFR-2 inhibitor), or IMC-18F1 (hVEGFR-1 inhibitor) or IMC-1121b and IMC-18F1. Data represent mean ± SE (n = 3). One-way ANOVA test was used. *, significantly different from control (BSA) p < 0.05; #, significantly different from rDLL4.

FIGURE 5.

DLL4-overexpressing cells induce endogenous DLL4 expression in the neighboring and adjacent cells DLL4 immunofluorescent labeling in pIRES-eGFP transfected HCMvECs versus pIRES-DLL4-eGFP transfected HCMvECs with or without L685,458. eGFP (green) signal allows to discriminate between transfected and nontransfected cells (A–C; GFP). pIRES-DLL4-eGFP transfected ECs revealed higher DLL4 staining (red) (B and C; DLL4) compared with pIRES-eGFP transfected cells (A; DLL4). Cells surrounding pIRES-eGFP-DLL4 transfected cells also showed increased DLL4 expression (B; DLL4) compare with cells surrounding pIRES-eGFP transfected cells (A; DLL4). This effect was prevented by adding the NOTCH signaling inhibitor L685,458 (C, DLL4). Merged images between eGFP and DLL4 staining (A–C; Merge) and phase contrast microscopy (PCI) images (A–C; PCI) are also reported. Scale bars, 50 μm.