Miki Yamazaki

Jagged1-selective Notch Signaling Induces Smooth Muscle Differentiation via a RBP-Jκ-dependent Pathway

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 SMC-myosin 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 byγ-secretase inhibitor, N-(N-(3,5-difluorophenyl)-l-alanyl)-S-phenylglycine t-butyl ester, which impedes Notch signaling. Using Rbp-jκ-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-Jκ, 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-Jκ-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.

HERP1 Inhibits Myocardin-Induced Vascular Smooth Muscle Cell Differentiation by Interfering With SRF Binding to CArG Box

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.

Runx2 represses myocardin-mediated differentiation and facilitates osteogenic conversion of vascular smooth muscle cells.

ABSTRACT Phenotypic plasticity and the switching of vascular smooth muscle cells (SMCs) play a critical role in atherosclerosis. Although Runx2, a key osteogenic transcription factor, is expressed in atherosclerotic plaques, the molecular mechanisms by which Runx2 regulates SMC differentiation remain unclear. Here we demonstrated that Runx2 repressed SMC differentiation induced by myocardin, which acts as a coactivator for serum response factor (SRF). Myocardin-mediated induction of SMC gene expression was enhanced in mouse embryonic fibroblasts derived from Runx2 null mice compared to wild-type mice. Forced expression of Runx2 decreased the expression of SMC genes and promoted osteogenic gene expression, whereas the reduction of Runx2 expression by small interfering RNA enhanced SMC differentiation in human aortic SMCs. Runx2 interacted with SRF and interfered with the formation of the SRF/myocardin ternary complex. Thus, this study provides the first evidence that Runx2 inhibits SRF-dependent transcription, as a corepressor independent of its DNA binding. We propose that Runx2 plays a pivotal role in osteogenic conversion tightly coupled with repression of the SMC phenotype in atherosclerotic lesions.

Notch signaling regulates the differentiation of bone marrow-derived cells into smooth muscle-like cells during arterial lesion formation.

Bone marrow- (BM-) derived cells can differentiate into smooth muscle-like cells (SMLC), resulting in vascular pathogenesis. However, the molecular mechanism of the differentiation remains unknown. We have recently reported that Notch signaling promotes while a Notch target HERP1 inhibit the differentiation of mesenchymal cells to SMC. During the differentiation of BM-derived mononuclear cells into smooth muscle alpha-actin (SMA)-positive cells, expression of Jagged1 and SMC-specific Notch3 was increased. Blocking Notch with gamma-secretase inhibitor prevented the induction of SMA. Wire-mediated vascular injury was produced in femoral arteries in mice transplanted with green fluorescent protein (GFP)-positive cells. Many double-positive cells for GFP/Jagged1 or GFP/Notch3 were detected in the thickened neointima. In contrast, only a few SMA-positive cells were positive for GFP in neointima where HERP1, a suppressor for Notch, were abundantly expressed. In conclusion, Notch-HERP1 pathway plays an important role in differentiation of BM-derived mononuclear cells into SMLC.

Homeobox Protein Hex Facilitates Serum Responsive Factor–Mediated Activation of the SM22α Gene Transcription in Embryonic Fibroblasts

Objective—Hex (hematopoietically expressed homeobox), a member of homeobox family of transcription factors, has been implicated in the vascular development because of its expression in hemangioblast, a hypothetical stem cell that gives rise to both angioblasts and hematopoietic lineages. In the present study, we examined the role of Hex in the differentiation of vascular smooth muscle cells. Methods and Results—We constructed adenovirus expressing Hex, to which we refer to as AxCA/Hex, and transduced murine embryonic fibroblasts, 10T1/2 cells. Northern blot analyses showed that Hex increased the mRNA levels of smooth muscle &agr;-actin and SM22&agr; but not of calponin and smooth muscle myosin heavy chain. Transient transfection assays showed that Hex activates the transcription from the SM22&agr; promoter in a CArG box-dependent manner. Electrophoretic mobility shift assays demonstrate that Hex is not able to bind to CArG box, but binding of serum responsive factor (SRF) to CArG box is enhanced in AxCA/Hex-transduced cells. Recombinant Hex protein produced by in vitro translation system augmented the binding activity of SRF to CArG box. Immunoprecipitation experiments revealed the physical association between Hex and SRF. Conclusions—Hex induces transcription of the SM22&agr; gene by facilitating the interaction between SRF and its cognate binding site in pluripotent embryonic fibroblasts.