Cheng Su-Li, Shao Jian-Su, Cai Jun, Sierra Oscar L, Towler Dwight A
Department of Medicine, Division of Bone and Mineral Diseases, Washington University School of Medicine, St Louis, MO 63110, USA.
J Biol Chem. 2008 Jul 18;283(29):20505-22. doi: 10.1074/jbc.M800851200. Epub 2008 May 15.
Msx2 is a homeodomain transcription factor first identified in craniofacial bone and human femoral osteoblasts. We hypothesized that Msx2 might activate skeletal Wnt signaling. Therefore, we analyzed the effects of CMV-Msx2 transgene (Msx2Tg) expression on skeletal physiology and composition. Skeletal Msx2 expression was increased 2-3-fold by Msx2Tg, with expanded protein accumulation in marrow, secondary ossification centers, and periosteum. Microcomputed tomography established increased bone volume in Msx2Tg mice, with increased numbers of plate-like trabeculae. Histomorphometry revealed increased bone formation in Msx2Tg mice versus non-Tg siblings, arising from increased osteoblast numbers. While decreasing adipogenesis, Msx2Tg increased osteogenic differentiation via mechanisms inhibited by Dkk1, an antagonist of Wnt receptors LRP5 and LRP6. Bone from Msx2Tg mice elaborated higher levels of Wnt7 canonical agonists, with diminished Dkk1, changes that augment canonical signaling. Analysis of non-Tg and Msx2Tg siblings possessing the TOPGAL reporter confirmed this; Msx2Tg up-regulated skeletal beta-galactosidase expression (p </= 0.01), along with Wnt7a and Wnt7b, and reduced circulating Dkk1. To better understand molecular mechanisms, we studied C3H10T1/2 osteoprogenitor cells. As in bone, Msx2 increased Wnt7 genes and down-regulated Dkk1, while inducing the osteoblast gene alkaline phosphatase. Msx2-directed RNA interference increased Dkk1 expression and promoter activity, while reducing Wnt7a, Wnt7b, and alkaline phosphatase. Moreover, Msx2 inhibited Dkk1 promoter activity and reduced RNA polymerase association with Dkk1 chromatin. RNA interference-mediated knockdown of Wnt7a, Wnt7b, and LRP6 significantly reduced Msx2-induced alkaline phosphatase. Msx2 exerts bone anabolism in part by reducing Dkk1 expression and enhancing Wnt signaling, thus promoting osteogenic differentiation of skeletal progenitors.
Msx2是一种同源结构域转录因子，最初在颅面骨和人类股骨成骨细胞中被鉴定出来。我们推测Msx2可能激活骨骼中的Wnt信号通路。因此，我们分析了CMV-Msx2转基因（Msx2Tg）表达对骨骼生理和组成的影响。Msx2Tg使骨骼中Msx2的表达增加了2至3倍，骨髓、次级骨化中心和骨膜中的蛋白质积累也有所增加。微型计算机断层扫描显示Msx2Tg小鼠的骨体积增加，板状小梁数量增多。组织形态计量学显示，与非转基因同胞相比，Msx2Tg小鼠的骨形成增加，这是由于成骨细胞数量增加所致。Msx2Tg在减少脂肪生成的同时，通过被Dkk1（Wnt受体LRP5和LRP6的拮抗剂）抑制的机制增加了成骨分化。Msx2Tg小鼠的骨骼产生了更高水平的Wnt7经典激动剂，同时Dkk1减少，这些变化增强了经典信号传导。对具有TOPGAL报告基因的非转基因和Msx2Tg同胞的分析证实了这一点；Msx2Tg上调了骨骼β-半乳糖苷酶的表达（p≤0.01），同时上调了Wnt7a和Wnt7b，并降低了循环中的Dkk1。为了更好地理解分子机制，我们研究了C3H10T1/2骨祖细胞。与在骨骼中的情况一样，Msx2增加了Wnt7基因的表达，下调了Dkk1的表达，同时诱导了成骨细胞基因碱性磷酸酶的表达。Msx2介导的RNA干扰增加了Dkk1的表达和启动子活性，同时降低了Wnt7a、Wnt7b和碱性磷酸酶的表达。此外，Msx2抑制了Dkk1启动子活性，并减少了RNA聚合酶与Dkk1染色质的结合。RNA干扰介导的Wnt7a、Wnt7b和LRP6的敲低显著降低了Msx2诱导的碱性磷酸酶。Msx2部分通过降低Dkk1表达和增强Wnt信号传导来发挥骨合成代谢作用，从而促进骨骼祖细胞的成骨分化。
