Abstract
Although previous studies demonstrate that appropriate Notch signaling is required during angiogenesis and in vascular homeostasis, the mechanisms by which Notch regulates vascular function remain to be elucidated. Here, we show that activation of the Notch pathway by the ligand Jagged1 reduces the proliferation of endothelial cells. Notch activation inhibits proliferation of endothelial cells in a cell-autonomous manner by inhibiting phosphorylation of the retinoblastoma protein (Rb). During cell cycle entry, p21Cip1 is upregulated in endothelial cells. Activated Notch inhibits mitogen-induced upregulation of p21Cip1 and delays cyclin D-cdk4-mediated Rb phosphorylation. Notch-dependent repression of p21Cip1 prevents nuclear localization of cyclin D and cdk4. The necessity of p21Cip1 for nuclear translocation of cyclin D-cdk4 and S-phase entry in endothelial cells was demonstrated by targeted downregulation of p21Cip1 by using RNA interference. We further demonstrate that when endothelial cells reach confluence, Notch is activated and p21Cip1 is downregulated. Inhibition of the Notch pathway at confluence prevents p21Cip1 downregulation and induces Rb phosphorylation. We suggest that Notch activation contributes to contact inhibition of endothelial cells, in part through repression of p21Cip1 expression.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at http://mcb.asm.org/.
We thank F. Wong and I. Pollet for technical help, R. E. Durand and D. McDougal for assistance with flow cytometry and cell sorting, and K. G. Leong for reviewing the manuscript.
This research was supported by grants to A.K. from the Heart and Stroke Foundation of British Columbia and Yukon, the National Cancer Institute of Canada with funds from the Canadian Cancer Society, and the Canadian Institutes of Health Research and to B.E.C. from the NIH (CA84069). M.N. was supported by a fellowship from Fondazione Italiana per la Ricerca sul Cancro, a fellowship from the Canadian Institutes of Health Research, and a Research Trainee Award from the Michael Smith Foundation for Health Research. G.M. was supported by a Research Trainee Award from the Michael Smith Foundation for Health Research. B.E.C. is a W. M. Keck Distinguished Young Scholar in Medical Research. A.K. is a Clinician-Scientist of the Canadian Institutes of Health Research and a Scholar of the Michael Smith Foundation for Health Research.