Abstract 350: Notch Signaling Accelerates Vascular Smooth Muscle Differentiation by Inducing Myocardin Gene Expression and by Enhancing the Transactivating Function of Myocardin.
Myocardin, a co-activator of serum response factor (SRF), is a major determinant of vascular smooth muscle cells (VSMC) differentiation. We have recently reported that Notch signaling, a crucial pathway for cell fate determination in various organs, positively and directly regulates VSMC differentiation in a RBP-Jκ-dependent manner as well. Here we test the hypothesis that Notch signaling regulates myocardin-mediated VSMC differentiation. RT-PCR analyses showed that overexpression of Notch1 intracellular domain (NICD), an activated form of Notch, strongly up-regulated myocardin gene expression in human aortic SMC, rat aortic SMC (RASMC) and 10T1/2 fibroblasts. In RASMC, the basal gene expression of myocardin as well as several SMC marker genes such as smooth muscle-myosin heavy chain (SM-MHC) and SM22α was markedly down-regulated by γ-secretase inhibitor, DAPT, which impedes Notch signaling. Luciferase reporter gene analyses showed that NICD transactivated 2-kb fragment of the mouse myocardin promoter containing a putative binding element for RBP-Jκ, an essential mediator of Notch signaling. Site directed mutagenesis revealed that the RBP-Jκ-binding site is indeed required for NICD-induced promoter activation. Gel shift assays and chromatin immunoprecipitation assays showed the binding of RBP-Jκ to its cognate binding sequence within the myocardin promoter. By using RBP-Jκ-deficient cells, we showed that NICD-induction of myocardin gene expression is dependent on RBP-Jκ protein. We further demonstrated that Notch signaling and myocardin synergistically promote VSMC differentiation. RT-PCR and luciferase reporter gene analyses showed that co-transfection of NICD- and myocardin-expression vectors remarkably enhanced transcription of the SM-MHC gene activated by either NICD or myocardin alone in 10T1/2 fibroblasts. Site directed mutagenesis of the binding sequences for either SRF or RBP-Jκ revealed that both sequences were necessary for the synergistic transactivation. Thus, we propose that Notch signaling plays a pivotal role in VSMC differentiation by inducing myocardin gene expression via a RBP-Jκ-dependent manner and by enhancing the ability of myocardin to activate the VSMC gene expression.