Abstract 502: Notch Signaling Enhances BMP-2-Mediated Osteogenic Conversion of Vascular Smooth Muscle Cells through a Synergistic Induction of Msx2 Gene Expression.
Bone morphogenetic protein2 (BMP2) and transcription factor Msx2, known as major regulators of osteoblast differentiation, have been implicated in vascular calcification. Recent evidences indicate that Notch signaling regulates valve calcification, atherosclerosis and osteogenesis. However, the role of Notch signaling in vascular calcification remains to be determined. In this study, we examined whether Notch signaling regulates BMP2-induced osteoblast differentiation of vascular smooth muscle cells (SMC) and mesenchymal cells. Treatment of either mouse mesenchymal 10T1/2 cells or human aortic smooth muscle cells (HASMC) with BMP2 induced the expression of the Msx2 genes as well as other osteoblast marker genes including ososteopontin and alkaline phosphatase (ALP). While forced expression of Notch intracellular domain (NICD) by adenovirus modestly induced Msx2 gene expression in the absence of BMP2, it markedly activated Msx2 gene expression as well as a set of osteoblast marker genes in the presence of BMP2. Cytochemical analyses using Alizarin red and von Kossa staining confirmed the osteogenic conversion of 10T1/2 cells transduced with adenovirus expressing NICD in the presence of BMP2. Expression of the Msx2 gene was induced in 10T1/2 cells by co-culture with fibroblast L-cells expressing Jagged1 or Dll4. Such an induction of the Msx2 gene expression was augmented by BMP2. Luciferase assay showed that transcription from the Msx2 promoter, which contains Smad binging elements (SBE), was synergistically activated by stimulation with Notch ligands and BMP2. Furthermore, this synergism was abrogated by mutation of SBE or gamma-secretase inhibitor DAPT, which blocks proteolytic process involved in Notch signaling. Immunohistochemistry of human calcifying atherosclerotic plaques revealed co-localization of Msx2, Cbfa1, Notch receptors and Notch ligands. In conclusions, these results suggest that BMP2 and Notch signaling synergistically promote a process by which vascular SMC and mesenchymal cells differentiate into osteoblast-like cells through a transcriptional activation of the Msx2 gene.