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Abstract
Notch signal transduction is evolutionarily conserved with essential functions identified in numerous species from C.elegans to H.Sapiens. Notch is critical during embryonic development, in the adult, and in cancer. In mammals there are four Notch receptors; much of our understanding of Notch signalling comes from the study of NOTCH1. During embryogenesis, Notch1 is widely expressed and is required in numerous processes including blood vessel formation and remodelling (angiogenesis). The view of Notch signal transduction, achieved primarily through studying NOTCH1, is that Notch receptors undergo processing and are transported to the cell surface as a heterodimer. Ligand binding triggers a series of proteolytic cleavages leading to the release of the Notch intracellular domain, which translocates to the nucleus to activate transcription. In contrast to NOTCH1, little is known about NOTCH4; Notch4 is expressed in endothelial cells of the circulatory system, trans-activation by ligand is difficult to detect, and a phenotype in mice, reported to be null for Notch4 (Notch4d1 allele), has not been identified. Here, further analysis of NOTCH4 has revealed many features that are in contrast to NOTCH1: (i) unprocessed NOTCH4 was present on the cell surface, (ii) ligand did not trans-activate NOTCH4 signalling, (iii) NOTCH4 inhibited NOTCH1 signal transduction in a dose dependent manner, (iv) NOTCH4 induced the differentiation of myoblasts, and (v) subcellular localisation of NOTCH4 was different to NOTCH1, and coexpression resulted in NOTCH1 adopting the NOTCH4 pattern of localisation. In addition, postnatal retinal angiogenesis was examined in mice carrying the Notch4d1 allele. A delay in the expansion of the retinal vasculature was equally observed in both Notch4d1+/- and Notch4d1-/- mice. This suggested that Notch4d1 was not a null allele. Fittingly, the Notch4d1 allele produced transcripts that encode for much of the extracellular domain of NOTCH4. The expression pattern of this transcript was equivalent to Notch4 in mouse embryos. Like full length NOTCH4, expression of a cDNA based on this transcript also inhibited NOTCH1 signal transduction. These studies reveal for the first time a function for NOTCH4 that is, as an inhibitor of NOTCH1. Accordingly it is postulated that Notch4 will have a crucial function in angiogenesis, since Notch1 is required for angiogenesis and Notch4 is expressed in endothelial cells of the circulatory system.