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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:1104.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

Notch Signaling Induces Osteogenic Differentiation and Mineralization of Vascular Smooth Muscle Cells

Role of Msx2 Gene Induction via Notch-RBP-Jk Signaling

Takehisa Shimizu; Toru Tanaka; Tatsuya Iso; Hiroshi Doi; Hiroko Sato; Keiko Kawai-Kowase; Masashi Arai; Masahiko Kurabayashi

From the Department of Medicine and Biological Science (T.S., T.T., T.I., H.D., H.S., K.K.-K., M.A., M.K.) and the Education and Research Center (T.I.), Gunma University Graduate School of Medicine, Japan.

Correspondence to Masahiko Kurabayashi, MD, PhD, Department of Medicine and Biological Science, Gunma University Graduate School of Medicine 3-39-15 Showa-machi, Maebashi, Gunma, 371-8511, Japan. E-mail mkuraba{at}med.gunma-u.ac.jp

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 induced osteogenic differentiation and matrix mineralization of HASMCs in vitro, in a manner dependent on the Msx2 gene, a master regulator of osteogenesis. Notch1, Jagged1, and Msx2 were expressed in human fibrocalcific/atherosclerotic plaques, suggesting that Notch–Msx2 signaling plays a important role in vascular calcification.

Key Words: Notch signaling • vascular smooth muscle cells • vascular calcification • Msx2