Notch4-induced inhibition of endothelial sprouting requires the ankyrin repeats and involves signaling through RBP-Jκ

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Abstract
Notch proteins comprise a family of transmembrane receptors. Ligand activation of Notch releases the intracellular domain of the receptor that translocates to the nucleus and regulates transcription through the DNA-binding protein RBP-Jκ. Previously, it has been shown that the Notch4 intracellular region (N4IC) can inhibit endothelial sprouting and angiogenesis. Here, N4IC deletion mutants were assessed for their ability to inhibit human microvascular endothelial cell (HMEC) sprouting with the use of a quantitative endothelial sprouting assay. Deletion of the ankyrin repeats, but not the RAM (RBP-Jκ associated module) domain or C-terminal region (CT), abrogated the inhibition of fibroblast growth factor 2 (FGF-2)- and vascular endothelial growth factor (VEGF)-induced sprouting by Notch4, whereas the ankyrin repeats alone partially blocked sprouting. The ankyrin repeats were also the only domain required for up-regulation of RBP-Jκ-dependent gene expression. Interestingly, enforced expression of the ankyrin domain alone was sufficient to up-regulate some, but not all, RBP-Jκ-dependent genes. Although N4IC reduced VEGF receptor-2 (VEGFR-2) and vascular endothelial (VE)-cadherin expression, neither of these events is necessary and sufficient to explain N4IC-mediated inhibition of sprouting. A constitutively active RBP-Jκ mutant significantly inhibited HMEC sprouting but not as strongly as N4IC. Thus, Notch4-induced inhibition of sprouting requires the ankyrin repeats and appears to involve RBP-Jκ-dependent and -independent signaling. (Blood. 2004;104:1760-1768)