Figure 1.
Intracellular domains of DLL4, DLL1 and JAG1 are highly conserved.
(A) ClustalW alignment of the intracellular domains of human DLL4, DLL1, and JAG1 with their respective homologues from mouse, cow, chicken, xenopus, zebrafish, and fugu revealed a high grade of conservation between all vertebrate species. The carboxyterminal PDZ motif (highlighted in yellow) is 100% conserved in all cases. (B) A dendrogram from a ClustalW alignment of the full-length proteins from vertebrates and drosophila proves that the proteins as a whole are also highly conserved between species. Colored boxes show the closer relationship of DLL1 and DLL4 compared to JAG1.
Figure 2.
JAG1 is processed by gamma-secretase.
HUVEC were transduced with adenoviruses expressing GFP, full length JAG1. The intracellular JAG1 domain fused to Citrine, or DLL1 tagged with V5 at the carboxyterminus. (A) JAG1 was processed and the cleavage products have the predicted size of the transmembrane-intracellular domain fragment (TM-ICD) and the free intracellular domain (ICD). The membrane was blotted with JAG1 antibodies recognizing the carboxyterminal intracellular domain. (B) Treatment with VEGF or FGF2 did not significantly increase ligand cleavage compared to solvent as control. Inhibition of gamma-secretase activity with 25 µM DAPT prevented the formation of the intracellular domain and led to accumulation of the uncleaved TM-ICD fragment. (C) The predicted size of the DLL1 intracellular domain, tagged with V5, was detected in HUVEC lysates by Western blotting.
Figure 3.
Localisation of Notch ligand intracellular domains.
(A) Scheme of the Notch ligand (DSL) intracellular domain (ICD) expression cassette in adenoviral vectors. Western blotting revealed expression of the fusion proteins in total lysates. (B) JAG1-ICD was localized predominantly in the nucleus, whereas DLL1-ICD and DLL4-ICD proteins were also localized in the cytoplasm. Scale bar, 100 µm. (C) Ligand-ICDs fused with Citrine were detected only in nuclear fractions (N) but not in the cytoplasmic fractions (C). HUVEC expressing full-length JAG1 or DLL1 fused to a c-terminal myc or V5-tag were lysed to obtain nuclear and cytoplasmic fractions. The processed intracellular fragments were detected only in the nuclear extracts by Western blotting.
Figure 4.
Notch ligand intracellular domains inhibit cellular proliferation but not migration and adhesion.
(A,B) Adenoviral expression of Delta and Jagged intracellular domains (ICD) in HUVEC inhibited BrdU incorporation indicating decreased cell proliferation. NOTCH1-ICD had stronger repression activity that was not altered by co-expression of DLL4-ICD or JAG1-ICD. n = 3 independent experiments. (C) Chemotactic migration of HUVEC through a transwell filter was not altered by ligand-ICDs but strongly impaired by NOTCH1-ICD expression. n = 3 independent experiments. (D) Adhesion of HUVEC to extracellular matrix proteins was enhanced by NOTCH1-ICD but not by ligand-ICDs. n = 3 independent experiments. Data are presented as mean +SD. *, p<0.05.
Figure 5.
Notch ligand ICDs do not affect sprouting angiogenesis.
NOTCH1-ICD or ligand-ICD expressing HUVEC were cultured as spheroids in collagen and treated with 25 ng/ml VEGF, FGF-2 or PBS as control. The cumulative length of all sprouts was measured. Data are presented as mean cumulative sprout length per spheroid +SD. n = 3 independent experiments with 10 spheroids per condition. (A,B) Expression of Citrine-tagged ligand-ICDs or such without this tag did not significantly alter sprouting behavior. (C) The average sprout length was slightly but not significantly (p>0.05 in all cases) reduced after ligand-ICD expression. The average number of capillary sprouts was not altered. (D) NOTCH1-ICD expression prevents VEGF and FGF2-induced sprouting angiogenesis. However, this was not significantly affected by co-expression of ligand-ICDs. *, p<0.05. n.s., not significant.
Figure 6.
JAG1-ICD does not interfere with NOTCH1-ICD in target gene induction.
HUVEC express only little amounts of HEY2 and HES5 mRNA. (A, B) Expression of NOTCH1-ICD induces strong expression of these genes 48 h after adenoviral transduction. This was not significantly affected by co-expression of JAG1, which itself had also no significant effects on mRNA expression of these endothelial Notch target genes. **, p<0.01. n = 3 independent experiments with 3 technical replicates.
Table 1.
Transcripts regulated by Notch ligand intracellular domains.