Molecular pathways of notch signaling in vascular smooth muscle cells

Abstract

Notch signaling in the cardiovascular system is important during embryonic development, vascular repair of injury, and vascular pathology in humans. The vascular smooth muscle cell (VSMC) expresses multiple Notch receptors throughout its life cycle, and responds to Notch ligands as a regulatory mechanism of differentiation, recruitment to growing vessels, and maturation. The goal of this review is to provide an overview of the current understanding of the molecular basis for Notch regulation of VSMC phenotype. Further, we will explore Notch interaction with other signaling pathways important in VSMC.

Keywords: Notch; signaling; vascular smooth muscle.

Figure 1

Overview of Notch signaling. Notch signaling is activated when a transmembrane ligand of the Delta–Serrate Lag (DSL) family (1) interacts with the EGF-like repeats of the extracellular domain of Notch receptors (2) on an adjacent cell. Ligand/receptor interactions induce a conformational change in the receptor, exposing critical sites for ADAM17 (S2) and γ-secretase (S3) cleavage of the Notch receptor (3). Cleavage of Notch receptors results in liberation and translocation of the intracellular domain (ICD) to the nucleus (4) while the extracellular domain (ECD) is endocytosed by the ligand-bearing cell (5). In the nucleus, the RAM domain of NotchICD binds to transcriptional repressor CBF1, causing displacement of associated co-repressors and recruitment of Mastermind-like (MAML) and other co-activators (6) to initiate transcription of Notch/CBF1 regulated downstream genes (7). Turning off Notch signaling begins with recruitment of CDK8 by MAML to the active complex, thereby causing phosphorylation of NotchICD within its PEST domain. Phosphorylation causes recruitment of E3 ubiquitin ligase Fbw7 for ubiquitination (8) and proteosome degradation of (9) of NotchICD, thereby destabilizing the activator complex and allowing CBF1 to re-associate with co-repressors.

Figure 2

Neonatal mice with smooth muscle-specific Jag-1 gene deletion exhibit patent ductus arteriosus. A conditional, Cre-responsive Jag-1 allele was crossed to the SM22α-Cre strain (TagIn-Cre) as described (Feng et al., 2010). To visualize the outflow tract and ductus arteriosus, the left ventricles of control littermate (A) and Jag-1 smooth muscle deletion (B) neonatal mice were injected with silicone rubber injection compound (tinted yellow). While the ductus arteriosus closed normally in the control mouse, it remained patent (open) in the Jag-1 smooth muscle deletion mouse. Associated defects in VSMC differentiation were also observed. DA, ductus arteriosus; LC, left carotid artery; LSC, left subclavian artery; RC, right carotid artery; RSC, right subclavian artery.

Figure 3

Jag-1/Notch3 signaling modulates VSMC plasticity toward maturation and contraction. Human aortic VSMC were plated on immobilized recombinant rat Jag-1 Fc or Fc control for 48 h to activate Notch receptors. Immunostaining reveals a significant increase in the expression of the contractile protein SM-actin in response to active Notch signaling (A). BrdU incorporation into the nuclei of Jag-1 Fc stimulated cells was significantly reduced in comparison to Fc control plated cells (B). (Green = BrdU positive nuclei, Blue = DAPI for total cell count, n = 15, p < 0.01). Reduction of Notch3 levels via siRNA-mediated knockdown (C) reveals a distinct reduction in endogenous SM-actin levels after 72 h as compared to VSMC receiving a non-targeting control probe (D).

Figure 4

Expression of Notch receptors in human arteries. Human lung biopsies were used for immunohistochemical analysis of Notch receptors expressed by medial VSMC cells. (A) Representative images of Notch receptors (green) and SM-actin positive cells (red) for Notch1 (left), Notch2 (middle), and Notch3 (right). (B) Images were overlaid with DAPI staining to show distribution and localization of Notch receptors within SM-actin positive cells. The elastic laminae display some autofluorescence.