Department of Oral & Cranio-maxillofacial Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology, No. 639 Zhizaoju Road, Shanghai, China.
Int J Oral Sci. 2019 Mar 18;11(2):12. doi: 10.1038/s41368-019-0046-1.
Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation, and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues. The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells. Initially, we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells. Moreover, Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line. In addition, micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo. Unexpectedly, Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2, Dlx5, Msx2, and Osterix, but led to upregulation of Alp and Osteocalcin (OCN), both of which play critical roles in promoting osteoblast maturation. Importantly, luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity. Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis, we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes. Based on these findings, we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene, suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.
遗传研究表明，远端同源盒（Dlx）基因在骨骼形成中起着关键作用，我们之前的研究表明，神经嵴细胞中 Dlx2 的过表达导致颅面组织的严重异常。本研究旨在探讨 Dlx2 在小鼠骨髓基质细胞（BMSCs）和成骨前体细胞 MC3T3-E1 细胞的成骨分化中的作用及其潜在的分子机制。最初，我们观察到 Dlx2 在 BMSCs 和 MC3T3-E1 细胞的早期成骨过程中上调。此外，Dlx2 的过表达增强了碱性磷酸酶（ALP）活性和 BMSCs 和 MC3T3-E1 细胞系的细胞外基质矿化。此外，裸鼠植入组织的微 CT 证实，BMSCs 中的 Dlx2 过表达促进了体内的骨形成。出乎意料的是，Dlx2 的过表达对关键成骨转录因子 Runx2、Dlx5、Msx2 和 Osterix 的表达水平几乎没有影响，但导致 Alp 和 Osteocalcin（OCN）的上调，这两者在促进成骨细胞成熟中起着关键作用。重要的是，荧光素酶分析表明，Dlx2 的过表达刺激了 OCN 和 Alp 启动子的活性。通过染色质免疫沉淀分析和定点突变分析，我们提供了分子证据，表明 Dlx2 通过直接结合这两个基因启动子中的 Dlx2 反应顺式作用元件，反式激活 OCN 和 Alp 的表达。基于这些发现，我们证明 Dlx2 过表达通过直接上调 OCN 和 Alp 基因增强体外成骨分化并加速体内骨形成，表明 Dlx2 在成骨分化和骨形成中起着关键作用。
