Abstract 5573: Notch Signaling Enhances BMP2-induced Msx2 Gene Expression and Vascular Calcification

Abstract

Background: Bone morphogenetic protein2 (BMP2) and transcription factor Msx2, known as major regulators of osteoblastic differentiation, have been implicated in the pathogenesis of vascular calcification. Recent evidences indicate that Notch signaling regulates valve calcification and atherosclerosis. In this study, we examined whether Notch signaling effects on BMP2-induced Msx2 gene expression and vascular calcification. Methods and Results: Treatment with either BMP2 or adenovirus expressing Notch intracellular domain (NICD), slightly induced the expression of the Msx2 gene in pluripotent mesenchymal stem cells and human aortic smooth muscle cells. Surprisingly, either NICD overexpression or stimulation by Notch ligands markedly augmented the Msx2 gene expression in the presence of BMP2, as well as other osteogenic genes expression. A knockdown of Msx2 expression using siRNA drastically attenuated synergistic ALP activity induced by BMP2 and NICD. Luciferase assay showed transcription activity of Msx2 promoter, which contains Smad binging elements (SBE) essential for BMP2 responsiveness, was synergistically enhanced by Notch ligands and BMP2 treatment, whereas this synergism was almost completely inhibited in the presence of Notch signaling blocker. Interestingly, DNA affinity precipitation assay revealed that NICD-RBPJK complex bound to Msx2 promoter very adjacently to SBE, which indicates that RBPJK-bound NICD might interact with Smads and this interaction might lead to enhanced BMP2 signaling and Msx2 expression. Correspondingly, OT11 cells, in which RBPJk expression was deficient, did not show increased Msx2 activation in response to BMP2 and Notch signaling. Conclusion: These results suggest that BMP2 and Notch signaling cooperatively promote osteogenic differentiation which may mediate vascular calcification in the development of atherosclerosis.