Dental and Craniofacial Research Institute, University of California Los Angeles, Los Angeles, United States of America.
PLoS One. 2011;6(9):e24638. doi: 10.1371/journal.pone.0024638. Epub 2011 Sep 13.
NELL-1 is a novel secreted protein associated with premature fusion of cranial sutures in craniosynostosis that has been found to promote osteoblast cell differentiation and mineralization. Our previous study showed that Runx2, the key transcription factor in osteoblast differentiation, transactivates the NELL-1 promoter. In this study, we evaluated the regulatory involvement and mechanisms of Osterix, an essential transcription factor of osteoblasts, in NELL-1 gene expression and function. Promoter analysis showed a cluster of potential Sp1 sites (Sp1/Osterix binding sites) within approximately 70 bp (from -71 to -142) of the 5' flanking region of the human NELL-1 transcriptional start site. Luciferase activity in our NELL-1 promoter reporter systems was significantly decreased in Saos-2 cells when Osterix was overexpressed. Mutagenesis study demonstrated that this suppression is mediated by the Sp1 sites. The binding specificity of Osterix to these Sp1 sites was confirmed in Saos-2 cells and primary human osteoblasts by EMSA in vitro and ChIP assay in vivo. ChIP assay also showed that Osterix downregulated NELL-1 by affecting binding of RNA polymerase II to the NELL-1 promoter, but not by competing with Runx2 binding to the OSE2 sites. Moreover, NELL-1 mRNA levels were significantly decreased when Osterix was overexpressed in Saos-2, U2OS, Hela and Glioma cells. Correspondingly, knockdown of Osterix increased NELL-1 transcription and osteoblastic differentiation in both Saos-2 cells and primary human osteoblasts. These results suggest that Osterix is a direct transcriptional regulator with repressive effect on NELL-1 gene expression, contributing to a delicate balance of regulatory effects on NELL-1 transcription with Runx2, and may play a crucial role in osteoblast differentiation and mineralization. These findings also extend our understanding of the molecular mechanism of Runx2, Osterix, and NELL-1 and demonstrate their crosstalk during osteogenesis.
NELL-1 是一种新型分泌蛋白，与颅缝早闭中颅缝过早融合有关，已发现其能促进成骨细胞分化和矿化。我们之前的研究表明，成骨细胞分化的关键转录因子 Runx2 可反式激活 NELL-1 启动子。在这项研究中，我们评估了成骨细胞必需转录因子 Osterix 在 NELL-1 基因表达和功能中的调节作用及其机制。启动子分析显示，在人类 NELL-1 转录起始位点的 5'侧翼区约 70bp（-71 至-142）内存在一个潜在的 Sp1 位点簇（Sp1/Osterix 结合位点）。当 Osterix 过表达时，我们的 NELL-1 启动子报告系统中的荧光素酶活性在 Saos-2 细胞中显著降低。突变研究表明，这种抑制是由 Sp1 位点介导的。在 Saos-2 细胞和原代人成骨细胞中，通过体外 EMSA 和体内 ChIP 测定证实了 Osterix 与这些 Sp1 位点的结合特异性。ChIP 测定还表明，Osterix 通过影响 RNA 聚合酶 II 与 NELL-1 启动子的结合而不是与 Runx2 竞争结合 OSE2 位点来下调 NELL-1。此外，当 Osterix 在 Saos-2、U2OS、Hela 和Glioma 细胞中过表达时，NELL-1mRNA 水平显著降低。相应地，在 Saos-2 细胞和原代人成骨细胞中敲低 Osterix 会增加 NELL-1 的转录和成骨分化。这些结果表明，Osterix 是一种直接的转录调节因子，对 NELL-1 基因表达具有抑制作用，有助于调节 Runx2 对 NELL-1 转录的调控作用达到微妙的平衡，并可能在成骨细胞分化和矿化中发挥关键作用。这些发现还扩展了我们对 Runx2、Osterix 和 NELL-1 分子机制的理解，并证明了它们在成骨过程中的相互作用。
