Hiroshi Doi

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.

Basic Fibroblast Growth Factor Antagonizes Transforming Growth Factor-β1–Induced Smooth Muscle Gene Expression Through Extracellular Signal–Regulated Kinase 1/2 Signaling Pathway Activation

Objective—Transforming growth factor-β1 (TGFβ1) and fibroblast growth factor (FGF) families play a pivotal role during vascular development and in the pathogenesis of vascular disease. However, the interaction of intracellular signaling evoked by each of these growth factors is not well understood. The present study was undertaken to examine the molecular mechanisms that mediate the effects of TGFβ1 and basic FGF (bFGF) on smooth muscle cell (SMC) gene expression. Methods and Results—TGFβ1 induction of SMC gene expression, including smooth muscle protein 22-&agr; (SM22&agr;) and smooth muscle &agr;-actin, was examined in the pluripotent 10T1/2 cells. Marked increase in these mRNA levels by TGFβ1 was inhibited by c-Src-tyrosine kinase inhibitors and protein synthesis inhibitor cycloheximide. Functional studies with deletion and site-directed mutation analysis of the SM22&agr; promoter demonstrated that TGFβ1 activated the SM22&agr; promoter through a CC(A/T-rich)6GG (CArG) box, which serves as a serum response factor (SRF)–binding site. TGFβ1 increased SRF expression through an increase in transcription of the SRF gene. In the presence of bFGF, TGFβ1 induction of SMC marker gene expression was significantly attenuated. Transient transfection assays showed that bFGF significantly suppressed induction of the SM22&agr; promoter–driven luciferase activity by TGFβ1, whereas bFGF had no effects on the TGFβ1-mediated increase in SRF expression and SRF:DNA binding activity. Mitogen-activated protein kinase kinase-1 (MEK1) inhibitor PD98059 abrogated the bFGF-mediated suppression of TGFβ1-induced SMC gene expression. Conclusion—Our data suggest that bFGF-induced MEK/extracellular signal-regulated kinase signaling plays an antagonistic role in TGFβ1-induced SMC gene expression through suppression of the SRF function. These data indicate that opposing effects of bFGF and TGFβ1 on SMC gene expression control the phenotypic plasticity of SMCs.

Enhancement of J–ST-Segment Elevation by the Glucose and Insulin Test in Brugada Syndrome

NOGAMI, A., et al.: Enhancement of J–ST‐Segment Elevation by the Glucose and Insulin Test in Brugada Syndrome. The effects of glucose and insulin on J–ST‐segment elevation were evaluated in seven men (mean age 45 ± 10 years) with Brugada syndrome. Six patients had been reanimated from VF and one patient had experienced syncope. The effects of intravenous (1) pilsicainide 50 mg, (2) glucose 50 g, and (3) glucose 50 g plus regular insulin 10 IU on the precordial ECG leads were examined. Pilsicainide significantly enhanced J‐ST elevation in all patients and induced VF in 1 patient. A significant accentuation of the abnormal J‐ST configuration was observed in all patients at a mean of 51 ± 40 minutes after glucose and insulin infusion. Changes in blood glucose and serum potassium concentration were 111 ± 158 mg/dL and −0.30 ± 0.48 mEq/L , respectively. These changes were not directly related to the ECG changes. Glucose infusion without insulin caused a subtle increase in J‐ST elevation. In conclusion, the administration of glucose and insulin safely unmasked or accentuation the J–ST‐segment elevation in Brugada syndrome. Blood glucose and insulin concentrations may be factors modulating the circadian or day‐to‐day ECG variations in this syndrome. (PACE 2003; 26[Pt. II]:332–337)

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 Induces Osteogenic Differentiation and Mineralization of Vascular Smooth Muscle Cells: Role of Msx2 Gene Induction via Notch-RBP-Jk Signaling

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 Msx2 gene, a key regulator of osteogenesis, was highly induced by coculture with Notch ligand-expressing cells or overexpression of Notch intracellular domains (NICDs) in human aortic SMCs (HASMCs). Furthermore, the Notch1 intracellular domain (N1-ICD) overexpression markedly upregulated alkaline phosphatase (ALP) activity and matrix mineralization of HASMCs. A knockdown experiment with a small interfering RNA confirmed that Msx2 mediated N1-ICD–induced osteogenic conversion of HASMCs. Interestingly, Msx2 induction by N1-ICD was independent of bone morphogenetic protein–2 (BMP-2), an osteogenic morphogen upstream of Msx2. The transcriptional activity of the Msx2 promoter was significantly enhanced by N1-ICD overexpression. The RBP-Jk binding element within the Msx2 promoter was critical to Notch-induced Msx2 gene expression. Correspondingly, N1-ICD overexpression did not induce the Msx2 expression in RBP-Jk–deficient fibroblasts. Immunohistochemistry of human carotid artery specimens revealed localization of Notch1, Jagged1 and Msx2 to fibrocalcific atherosclerotic plaques. Conclusion—These results imply a new mechanism for osteogenic differentiation of vascular SMCs in which Notch/RBP-Jk signaling directly induces Msx2 gene expression and suggest its crucial role in mediating vascular calcification.

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.

Molecular cloning of human epidermal transglutaminase cDNA from keratinocytes in culture.

We have isolated a cDNA encoding human epidermal transglutaminase, a key enzyme of terminal differentiation of keratinocytes. A cDNA library from cultured human keratinocytes was screened by a PCR-amplified partial cDNA fragment of the enzyme with oligonucleotide primers based on the homology of the transglutaminase family. The cDNA is 2734 bp coding a protein of 817 amino acids. The several regions including the active site cysteine residue are highly conserved among the transglutaminase family. However, the charged N-terminal domain is unique to the epidermal transglutaminse, suggesting that the region is involved in the function of the enzyme in keratinocytes.