The Notch signaling pathway plays a crucial role in specifying cellular fates by interaction between cellular neighbors; however, the molecular mechanism underlying smooth muscle cell (SMC) differentiation by Notch signaling has not been well characterized. Here we demonstrate that Jagged1-Notch signaling promotes SMC differentiation from mesenchymal cells. Overexpression of the Notch intracellular domain, an activated form of Notch, up-regulates the expression of multiple SMC marker genes including SMCmyosin heavy chain (Sm-mhc) in mesenchymal 10T1/2 cells, but not in non-mesenchymal cells. Physiological Notch stimulation by its ligand Jagged1, but not Dll4, directly induces Sm-mhc expression in 10T1/2 cells without de novo protein synthesis, indicative of a ligand-selective effect. Jagged1-induced expression of SM-MHC was blocked by ␥-secretase inhibitor, N-(N-(3,5-difluorophenyl)-Lalanyl)-S-phenylglycine t-butyl ester, which impedes Notch signaling. Using Rbp-j-deficient cells and site-specific mutagenesis of the SM-MHC gene, we show that such an induction is independent of the myocardin-serum response factor-CArG complex, but absolutely dependent on RBP-J, a major mediator of Notch signaling, and its cognate binding sequence. Of importance, Notch signaling and myocardin synergistically activate SM-MHC gene expression. Taken together, these data suggest that the Jagged1-Notch pathway constitutes an instructive signal for SMC differentiation through an RBP-J-dependent mechanism and augments gene expression mediated by the myocardin-SRF-CArG complex. Given that Notch pathway components are expressed in vascular SMC during normal development and disease, Notch signaling is likely to play a pivotal role in such situations to modulate the vascular smooth muscle cell phenotype. Vascular smooth muscle cells (VSMC)2 maintain considerable phenotypic plasticity throughout life and this plasticity is essential for vascular development and the pathogenesis of vascular disease such as atherosclerosis and restenosis following angioplasty (1). Differentiated VSMC exhibit a contractile phenotype that is characterized by a low rate of proliferation, low synthetic activity, and expression of a unique repertoire of proteins such as smooth muscle ␣-actin (SM ␣-actin), SM22␣, h-caldesmon (h-CaD), smoothelin-B, and smooth muscle-myosin heavy chain (SM-MHC). Once VSMC are exposed to injurious stimuli, they dedifferentiate into a so-called synthetic phenotype that exhibits a high rate of proliferation, high synthetic activity, and down-regulated expression of SMC marker genes accompanied by the induction of some marker genes for immature SMC.Among the many transcription factors involved in SMC differentiation, the myocardin-SRF complex is a key regulator as its expression is essential for SMC differentiation (2-5). Promoter/enhancer regions of most SMC marker genes such as SM-MHC and SM22␣ include several SRF binding elements, referred to as CArG or SRE. SRF recruits a potent coactivator, myocardin, resulting in the induction of SMC...
Objective-Myocardin is a coactivator of serum response factor (SRF) required for vascular smooth muscle cell (VSMC) differentiation. HERP1 is a transcriptional repressor, which is abundantly expressed in vascular system and is known to function as a target gene of Notch. However, the role of HERP1 in the pathogenesis of vascular lesions remains unknown. The present study characterizes the expression of HERP1 in normal and diseased vessels, and tests the hypothesis that HERP1 inhibits SRF/myocardin-dependent SMC gene expression. Methods and Results-Immunohistochemistry revealed that HERP1 and myocardin expression was localized to SMC in the neointima of balloon-injured rat aorta and in human coronary atherosclerotic lesions. Expression of both HERP1 and myocardin was elevated in cultured VSMCs compared with medial SMC. Overexpressed HERP1 inhibited the myocardin-induced SMC marker gene expression in 10T1/2 cells. HERP1 protein interfered with the SRF/CArG-box interaction in vivo and in vitro. Immunoprecipitation assays showed that HERP1 physically interacts with SRF. Conclusions-HERP1 expression was associated with the SMC proliferation and dedifferentiation in vitro and in vivo.HERP1 may play a role in promoting the phenotypic modulation of VSMCs during vascular injury and atherosclerotic process by interfering with SRF binding to CArG-box through physical association between HERP1 and SRF. Key Words: HERP1 Ⅲ myocardin Ⅲ serum response factor Ⅲ smooth muscle cells P henotypic modulation of vascular smooth muscle cells (VSMCs) from contractile to synthetic forms plays a pivotal role in the pathogenesis of vascular diseases including atherosclerosis and restenosis after angioplasty. 1 It is wellestablished that VSMC phenotype is regulated by a complex array of local environmental cues including humoral factors, cell-cell and cell-matrix interactions, inflammatory stimuli, and mechanical stresses. Such complex stimuli downregulate a number of genes required for the contractile phenotype in synthetic VSMCs. These include smooth muscle myosin heavy chain (SM-MHC), SM22␣, caldesmon, and calponin. Because the genes encoding these proteins are differentially expressed depending on the proliferative state of VSMCs, transcription factors regulated by numerous stimuli are responsible at least in part for the distinct pattern of gene expression seen in synthetic VSMCs.There is mounting evidence that most SMC marker proteins such as SM-MHC and SM22␣ are controlled by serum response factor (SRF), which binds to a sequence known as a CArG box and recruits a potent coactivator, myocardin, for SMC differentiation. 1 When myocardin is ectopically expressed in nonmuscle cells, it can induce SMC differentiation. 2,3 Most importantly, mouse embryos deficient for myocardin show no evidence of vascular SMC, indicating myocardin as a necessary and sufficient factor for SMC differentiation in vivo. 4 These observations, in conjunction with downregulation of SMC marker genes in synthetic VSMC, led us to speculate that myocardin express...
Objective-Vascular calcification is closely correlated with cardiovascular morbidity and mortality. Here, we demonstrate the role of Notch signaling in osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs). Methods and Results-The
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