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).


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.


CONCLUSIONS
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.
Shimizu
Takehisa
T
Department of Medicine and Biological Science, Gunma University Graduate School of Medicine 3-39-15 Showa-machi, Maebashi, Gunma, 371-8511, Japan.
Tanaka
Toru
T
Iso
Tatsuya
T
Doi
Hiroshi
H
Sato
Hiroko
H
Kawai-Kowase
Keiko
K
Arai
Masashi
M
Kurabayashi
Masahiko
M
D001011:Aorta / Q000166:cytology; D002454:Cell Differentiation / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D051880:Receptors, Notch / Q000502:physiology; D015398:Signal Transduction; D015533:Transcriptional Activation
D018398:Homeodomain Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C084119:MSX2 protein; D051880:Receptors, Notch