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, 293 (4), 1229-1242

Control of Endothelial Cell Tube Formation by Notch Ligand Intracellular Domain Interactions With Activator Protein 1 (AP-1)

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Control of Endothelial Cell Tube Formation by Notch Ligand Intracellular Domain Interactions With Activator Protein 1 (AP-1)

Zary Forghany et al. J Biol Chem.

Abstract

Notch signaling is a ubiquitous signal transduction pathway found in most if not all metazoan cell types characterized to date. It is indispensable for cell differentiation as well as tissue growth, tissue remodeling, and apoptosis. Although the canonical Notch signaling pathway is well characterized, accumulating evidence points to the existence of multiple, less well-defined layers of regulation. In this study, we investigated the function of the intracellular domain (ICD) of the Notch ligand Delta-like 4 (DLL4). We provide evidence that the DLL4 ICD is required for normal DLL4 subcellular localization. We further show that it is cleaved and interacts with the JUN proto-oncogene, which forms part of the activator protein 1 (AP-1) transcription factor complex. Mechanistically, the DLL4 ICD inhibited JUN binding to DNA and thereby controlled the expression of JUN target genes, including DLL4 Our work further demonstrated that JUN strongly stimulates endothelial cell tube formation and that DLL4 constrains this process. These results raise the possibility that Notch/DLL4 signaling is bidirectional and suggest that the DLL4 ICD could represent a point of cross-talk between Notch and receptor tyrosine kinase (RTK) signaling.

Keywords: AP1 transcription factor (AP-1); Notch pathway; angiogenesis; gene regulation; signaling.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
A, the ICD of DLL4 is evolutionarily conserved. Highlighted are the transmembrane domain (underlined), a putative caspase cleavage site (red), and a PDZ-binding motif (green). *, conserved amino acids; :, partly conserved amino acids (different amino acids are similar); ., partly conserved amino acids (different amino acids are not similar). B, the DLL4 ICD is necessary for appropriate DLL4 subcellular localization, and untethered DLL4INTRA is enriched in the nucleus. HUVECs were infected with lentiviruses for stably expressing the indicated HA epitope–tagged DLL4 constructs. DLL4N lacks the ICD and is otherwise identical to wildtype DLL4. DLL4INTRA encompasses the ICD alone. The subcellular distribution of DLL4 was visualized by immunofluorescence using an HA antibody. Scale bar, 20 μm. C–E, the ICD of DLL4 is cleaved. C, mass spectrometry of cleaved DLL4 fragments. Endogenous DLL4 or DLL4 expressing a C-terminal HA epitope tag was immunopurified from HUVECs. Samples were separated by SDS-PAGE, and DLL4 ICDs were subjected to in-gel digestion with trypsin followed by on-line nanoflow LC-MS/MS analysis of the peptide mixture on an LTQ Orbitrap Velos mass spectrometer. The MS/MS spectrum of a recovered peptide corresponding to amino acids 608–624 of the DLL4 ICD is shown. D, the indicated ICD methionine residues of HA epitope–tagged full-length DLL4 were mutated to alanine. HA antibody Western blotting was performed on lysates prepared from HUVECS stably expressing the indicated DLL4 ligands. E, a conserved LEVD motif is required for cleavage of the DLL4 ICD. Left panel, the indicated nested deletions of HA epitope–tagged full-length DLL4 were generated by site-directed mutagenesis. HA antibody Western blotting was performed on lysates prepared from HUVECs stably expressing the indicated DLL4 ligands. Corresponding deletions are highlighted above. Right panel, DLL4 ICD cleavage is blocked by the pan-caspase inhibitor Z-VAD but not DAPT. Cells expressing wildtype HA epitope–tagged full-length DLL4 were incubated overnight with or without the indicated concentrations of Z-VAD or DAPT. DLL4 protein species were detected by HA antibody Western blotting. Ab, antibody; DLL4FL, full-length DLL4.
Figure 2.
Figure 2.
DLL4INTRA interacts with the bZIP domain of JUN. A, yeast two-hybrid baits were constructed by fusing DLL4 ICD fragments in-frame with a LexA DNA-binding domain either at the N or the C terminus of the ICD. Constructs were expressed in yeast to verify expression by Western blotting of lysates using a LexA antibody. A p53-LexA fusion is included as a control (Dualsystems Biotech). Predicted full-length proteins of constructs used to screen a library prepared from HUVECs are marked with an asterisk. B, a PLA revealed an endogenous DLL4-JUN complex. The graph shows relative complex formation per cell (right upper panel) that was abolished by ablation of either DLL4 or JUN (siRNA efficacies were demonstrated using cells ectopically expressing either DLL4 or JUN; see right lower panels). The endogenous DLL4-JUN complex was undetectable using single antibodies alone. Quantification was performed as described (see “Experimental procedures”) with an average of 100 cells scored. Scale bar, 20 μm. C, JUN specifically associated with the ICD of DLL4. PLA was performed on HUVECs ectopically expressing HA epitope–tagged versions of either full-length DLL4 (DLL4FL); DLL4N, which lacks the ICD but retains the transmembrane domain; or the non-membrane–tethered DLL4INTRA. Relative protein levels of DLL4 and endogenous JUN were determined by Western blotting with the indicated antibodies (right lower panel). The DLL4-JUN complex was detected using a combination of HA (for DLL4) and endogenous JUN antibodies. The graph shows relative complex formation per cell (right upper panel). Quantification was performed as described (see “Experimental procedures”) with an average of 100 cells scored. Scale bar, 20 μm. D and E, the ICD of DLL4 interacts biochemically with the bZIP domain of JUN. D, recombinant bZIP domains of JUN and CREB3 (and a control protein consisting of the DNA-binding domain of the TEL/ETV6 ETS transcription factor) were incubated with in vitro translated HA epitope–tagged DLL4INTRA. Recombinant proteins were visualized by Coomassie staining. Bound DLL4INTRA was detected by Western blotting. E, recombinant JUN domains (and a control protein) were incubated with in vitro translated HA epitope–tagged full-length DLL4, DLL4N, or DLL4INTRA. Recombinant proteins were visualized by Coomassie staining. Bound DLL4 was detected by Western blotting. F, the C terminus of DLL4INTRA is necessary for binding of DLL4INTRA to the JUN bZIP domain. Experiments were performed as in D and E. DLL4INTRAΔN lacks the N-terminal amino acids 553–567. DLL4INTRAΔC lacks the C-terminal amino acids 666–686. G, the indicated constructs were stably expressed in HUVECs. JUN-DLL4 complexes were immunopurified from cell lysates using a rabbit HA polyclonal antibody and visualized by Western blotting with a FLAG mouse monoclonal antibody. TM, transmembrane domain; IP, immunoprecipitation; TL, total lysate.
Figure 3.
Figure 3.
DLL4INTRA antagonizes JUN DNA binding. A, JUN binds to a consensus AP-1 DNA-binding site in vitro. A biotinylated double-stranded oligonucleotide harboring three consensus AP-1–binding sites was incubated with the indicated FLAG epitope–tagged in vitro translated proteins. JUNΔDBD lacks the DNA-binding domain. JUNΔC(309–331) lacks the C-terminal amino acids abutting the DNA-binding domain. DNA-bound JUN was detected by Western blotting. B, DLL4INTRA impedes JUN binding to DNA. As in A, AP-1 DNA–binding sites were incubated with the indicated in vitro translated proteins. DNA-bound JUN was detected by Western blotting. Right panel, in vitro translated FLAG epitope–tagged JUN, untagged FOS, and HA epitope–tagged INTRA were incubated as shown in the absence of the AP-1 site. FLAG epitope–tagged JUN was immunopurified on FLAG beads, and associated complexes were detected by Western blotting. C, the putative PDZ-binding motif of DLL4INTRA is dispensable for DLL4INTRA binding to JUN. As in A and B, AP-1 DNA-binding sites were incubated with the indicated in vitro translated proteins. INTRAΔ(ATEV) lacks the putative C-terminal PDZ-binding motif. INTRAΔN(553–593) lacks the highlighted N-terminal amino acids. INTRAΔ(666–686) lacks the C-terminal 20 amino acids. DNA-bound JUN was detected by Western blotting. D, DLL4INTRA alters the JUN-controlled transcriptome. A microarray analysis was performed on HUVECs stably expressing the indicated constructs. Protein levels were determined by Western blotting with the indicated antibodies (lower panel). Heat maps show a comparison of global gene expression profiles (upper panels). E, expression levels in HUVECs of the indicated transcripts were determined by real-time qPCR. All values were averaged relative to TBP, SRPR, and CAPNS1. Values were normalized against mock-treated cells. Values represent ±S.D. (n = 3). F, JUN controls DLL4 expression. Real-time qPCR was performed on cDNA prepared from cells stably infected with one of four different JUN shRNA constructs and stimulated with or without VEGF for the shown time course (minutes). All values were averaged relative to TBP, SRPR, and CAPNS1. Values were normalized against the time 0 time point of mock-infected cells. Values represent ±S.D. (n = 3). G and H, JUN associates with the proximal promoter of human DLL4. G, upper panel, a ChIP analysis was performed on HUVECs incubated with or without 50 ng/ml VEGF for the indicated times. Three different primer sets centered on the illustrated promoter region were used, and a single representative is shown (all three gave very similar results). Equivalent amounts of rabbit IgG were used as a control, and results are presented as -fold changes in recovery (as a fraction of input) relative to the zero time point. Lower panel, expression of a stably integrated luciferase reporter was placed under the control of the depicted wildtype DLL4 proximal promoter or the same promoter in which the putative AP-1 sites have been singly or doubly mutated. The alignment of the human and mouse DLL4 promoter regions highlights the presumed transcription start site (underlined). Conserved consensus AP-1 DNA-binding sites are shown in green. A ChIP analysis was performed on reporter-expressing HUVECs incubated for 30 min with 50 ng/ml VEGF. Equivalent amounts of rabbit IgG were used as a control, and results are presented as -fold changes in recovery (as a fraction of input) relative to the control IgG antibody. Two different primer sets were centered on the integrated luciferase gene. A single representative is shown (both gave comparable results). H, upper graphs, ChIP analyses were performed with the indicated antibodies (as described in G) on control HUVECs and HUVECs stably expressing DLL4INTRA. Lower graphs, DLL4 expression levels in control HUVECs and HUVECs stably expressing DLL4INTRA were determined by real-time qPCR. All values were averaged relative to TBP, SRPR, and CAPNS1. Values were normalized against mock-treated cells. Values represent ±S.D. (n = 3). IP, immunoprecipitation; VWF, von Willebrand factor. Error bars represent S.D.
Figure 4.
Figure 4.
A, JUN strongly stimulates endothelial cell tube formation/sprouting. HUVECs lacking endogenous JUN or ectopically expressing the indicated JUN proteins were cultured in Matrigel in the presence of 50 ng/ml VEGF. A representative of several independent experiments is shown. After 24 h, in-house computer software was used to quantify the total length of the sprouts, the number of branch points, and the number of loops. Protein levels were determined by Western blotting with the indicated antibodies. Scale bar, 500 μm. B, DLL4INTRA attenuates JUN-mediated sprouting. HUVECs ectopically expressing the indicated proteins were cultured in Matrigel in the presence of 50 ng/ml VEGF. Experiments were conducted and quantified as in A. Scale bar, 500 μm. DBD, DNA-binding domain; DLL4FL, full-length DLL4.
Figure 5.
Figure 5.
A, JUNB strongly stimulates endothelial cell tube formation/sprouting. HUVECs ectopically expressing JUN or JUNB were cultured in Matrigel in the presence of 50 ng/ml VEGF. A representative of several independent experiments is shown. After 48 h, in-house computer software was used to quantify the total length of the sprouts, the number of branch points, and the number of loops. Protein levels were determined by Western blotting with the indicated antibodies. Scale bar, 500 μm. B, DLL4INTRA attenuates JUNB-mediated sprouting. HUVECs ectopically expressing the indicated proteins were cultured in Matrigel in the presence of 50 ng/ml VEGF. A representative of several independent experiments is shown. After 24 h, in-house computer software was used to quantify the total length of the sprouts, the number of branch points, and the number of loops. Protein levels were determined by Western blotting with the indicated antibodies. Scale bar, 500 μm.

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