Notch Signaling Pathway Enhances Bone Morphogenetic Protein 2 (BMP2) Responsiveness of Msx2 Gene to Induce Osteogenic Differentiation and Mineralization of Vascular Smooth Muscle Cells

Abstract Vascular calcification is regulated in a process similar to bone formation. BMP2 (bone morphogenetic protein 2) is essential for osteoblastic differentiation of mesenchymal progenitor cells and thus has been implicated in the development of vascular calcification. Here we examined whether Notch signaling interacts with BMP2 signaling to regulate osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs). BMP2 alone scarcely induced the expression of alkaline phosphatase (ALP), an ectoenzyme crucially required for active biomineralization, in human aortic SMCs (HASMCs), despite its strong induction in osteoblast precursor MC3T3-E1 cells. Notably, overexpression of the Notch1 intracellular domain (N1-ICD) markedly enhanced BMP2-mediated induction of ALP activity and mineralization of HASMCs. In HASMCs, expression of Msx2 gene, a well documented BMP2 target gene in osteoblasts, was barely induced by BMP2 alone, and N1-ICD clearly enhanced the BMP2-driven Msx2 gene expression. Deletion and site-directed mutation analysis of Msx2 gene promoter revealed that the RBPJk-binding site was necessary for BMP2 responsiveness. Using the RBPJk-deficient cells and siRNA for RBPJk, we showed that RBPJk was required for BMP2 induction of Msx2 gene expression and ALP activity. Moreover, we showed that Smad1, a transcription factor downstream of BMP2 signaling, interacted with N1-ICD to form a complex within the Msx2 promoter. Immunohistochemistry of human calcifying atherosclerotic plaques revealed colocalized expression of Notch1, BMP2, and Msx2. These results indicate that the Notch intracellular domain·RBPJk complex enhances the BMP2-induced Msx2 gene expression by cooperating with Smad1 and suggest that Notch signaling makes vascular SMC responsive to BMP2 and promotes vascular calcification.