Department of Medicine and Biological Science, Gunma University Graduate School of Medicine, Gunma 371-8511, Japan.
J Biol Chem. 2011 May 27;286(21):19138-48. doi: 10.1074/jbc.M110.175786. Epub 2011 Apr 6.
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.
血管钙化受类似于骨形成的过程调控。BMP2（骨形态发生蛋白 2）对于间充质祖细胞的成骨细胞分化是必不可少的，因此与血管钙化的发生有关。在这里，我们研究了 Notch 信号是否与 BMP2 信号相互作用，以调节血管平滑肌细胞（VSMCs）的成骨分化和矿化。尽管 BMP2 在成骨前体细胞 MC3T3-E1 细胞中强烈诱导，但 BMP2 单独很少诱导碱性磷酸酶（ALP）的表达，ALP 是主动生物矿化所必需的外切酶，在人主动脉平滑肌细胞（HASMCs）中。值得注意的是，Notch1 细胞内结构域（N1-ICD）的过表达显着增强了 BMP2 介导的 HASMCs 中 ALP 活性和矿化的诱导。在 HASMCs 中，BMP2 单独作用几乎不能诱导 Msx2 基因的表达，而 Msx2 基因是成骨细胞中众所周知的 BMP2 靶基因，N1-ICD 明显增强了 BMP2 驱动的 Msx2 基因表达。Msx2 基因启动子的缺失和定点突变分析表明，RBPJk 结合位点对于 BMP2 反应性是必需的。使用 RBPJk 缺陷细胞和针对 RBPJk 的 siRNA，我们表明 RBPJk 是 BMP2 诱导 Msx2 基因表达和 ALP 活性所必需的。此外，我们表明 BMP2 信号下游的转录因子 Smad1 与 N1-ICD 相互作用，在 Msx2 启动子内形成复合物。对人钙化动脉粥样硬化斑块的免疫组织化学分析显示 Notch1、BMP2 和 Msx2 的共表达。这些结果表明，Notch 细胞内结构域·RBPJk 复合物通过与 Smad1 合作增强了 BMP2 诱导的 Msx2 基因表达，并表明 Notch 信号使血管平滑肌细胞对 BMP2 有反应并促进血管钙化。
