Determinants of Notch-3 receptor expression and signaling in vascular smooth muscle cells. Implications in cell-cycle regulation

Authors:
Campos AH, Wang W, Pollmann MJ and Gibbons GH
In:
Circ Res (2002) 91(11): 999-1006
Research Area:
Cardiovascular
Platform:
Nucleofectorâ„¢ I/II/2b
Experiment
The Notch family of receptors and ligands plays an important role in cell fate determination, vasculogenesis, and organogenesis. However, the expression and regulation of the Notch signaling pathway remains to be defined. The ligands Jagged and Delta can bind to various members of the Notch receptor family and promote activation by inducing proteolytic cleveage of the intracellular domain (IC) of Notch. In certain contexts, the IC of the Notch receptor translocates to the nucleus and converts the transcription factor CBF-1 from a repressor to an activator of target gene expression. To further reveal the role of CBF-1, the rat aortic smooth muscle cell line A7r5 was nucleofected with luciferase reporter plasmids encoding wild type (WT) or mutated CBF-1. Cells transfected with the mutated CBF-1 showed a 2 fold increase in luciferase activity compared to the WT CBF-1. Together with other findings the results suggest that the pathogenesis of vascular disease may involve alterations in cell proliferation that are governed by the activity of the Notch-3 receptor-signaling pathway.
Abstract
The Notch family of receptors and ligands plays an important role in cell fate determination, vasculogenesis, and organogenesis. Mutations of the Notch-3 receptor result in an arteriopathy that predisposes to early-onset stroke. However, the functional role of the Notch signaling pathway in adult vascular smooth muscle cells (VSMCs) is poorly characterized. This study documents that the Notch-3 receptor, the ligand Jagged-1, and the downstream transcription factor, HESR-1, are expressed in the normal adult rat carotid artery, and that this expression is modulated after vascular injury. In cultured VSMCs, both angiotensin II and platelet-derived growth factor (PDGF) markedly downregulated Notch-3 and Jagged-1 through ERK-dependent signaling mechanisms and prevented the glycosylation of Jagged-1. The downregulation of Jagged-1 and Notch-3 was associated with a decrease in CBF-1-mediated gene transcription activation and a fall in the mRNA levels of the downstream target transcription factor HESR-1. To test the hypothesis that the Notch pathway was coupled to growth regulation, we generated VSMC lines overexpressing the constitutively active form of Notch-3 (A7r5-N3IC). These cells exhibited a biphasic growth behavior in which the growth rate was retarded during the subconfluent phase and failed to decelerate at postconfluence. The lack of cell-cycle arrest in postconfluent A7r5-N3IC was associated with an attenuated upregulation of the cell-cycle inhibitor p27(kip) relative to control cells. This study documents the regulation of the Jagged-1 and Notch-3 genes in VSMCs by growth factor stimulation as well as a role for Notch-3 as a determinant of VSMC growth.