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Mechanisms of Notch4-induced inhibition of endothelial sprouting Mackenzie, Farrell Ian


Notch proteins comprise a family of transmembrane receptors. Ligand activation of Notch releases an intracellular region of the receptor that translocates to the nucleus and regulates transcription through interaction with the DNA-binding protein CBF1. CBF1- independent Notch signalling has also been described. Previously, the human Notch4 intracellular region (N4IC) was shown to inhibit endothelial sprouting in vitro and angiogenesis in vivo. The objective of this study was to determine the N4IC domains required for inhibition of endothelial sprouting and to resolve whether this inhibition involves CBFl-dependent signalling. N4IC contains a RAM domain and six ankyrin repeats for protein binding and a Cterminal region (CT) that has poorly defined function. The necessity of each domain for Notch4 activity in endothelial cells was analysed using human dermal microvascular endothelial cells (HMEC-1) expressing N4IC or N4IC deletion constructs. As shown by immunofluorescent staining, mutants lacking the RAM domain had reduced nuclear localisation. Nuclear targeting was restored by fusion of a viral nuclear localisation signal. The angiogenic effect of the N4IC mutants was quantitated using an in vitro endothelial sprouting assay. Deletion of the ankyrin domain, but not the RAM or CT, abrogated the inhibition of FGF-2- and VEGF-induced sprouting, while the ankyrin repeats alone partially blocked sprouting. N4IC decreased VEGF Receptor 2 (VEGFR2) mRNA expression but did not alter FGF Receptor 1 (FGFR1) levels. Conversely, constructs lacking the RAM or CT increased VEGFR2 mRNA compared to controls. Therefore, Notch4 appears to block endothelial sprouting through mechanisms other than mere downregulation of VEGFR2 and FGFR1. The ankyrin repeats, but not the RAM or CT, were also required for upregulation of CBFl-dependent gene expression as shown by luciferase reporter assays and RT-PCR for endogenous CBF1 targets. The ankyrin domain alone was sufficient to upregulate some, but not all, CBFl-dependent genes. Fusion of CBF1 to a viral transactivation domain created a construct that induced expression of target genes independently of N4IC. This constitutively-active CBF1 significantly inhibited HMEC-1 sprouting, but not as strongly as N4IC. Therefore, the inhibition of endothelial sprouting by N4IC requires the ankyrin repeats and appears to involve CBFl-dependent and -independent signalling.

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