Department of Endodontics, School of Stomatology, China Medical University, Nanjing North Street 117, Shenyang, 110002, China.
Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata, Kitaku, Okayama, 770-8525, Japan.
Cell Biol Int. 2018 Apr;42(4):403-410. doi: 10.1002/cbin.10902. Epub 2017 Nov 15.
Serine/threonine protein phosphatase 2A (PP2A) is involved in regulating various physiological processes including cell cycle, growth, apoptosis, and signal transduction. Osteoblast differentiation is controlled by main bone specific transcription factors including Osterix, distal-less homeobox 5 (Dlx5), and Runt-related transcription factor 2 (Runx2). We previously reported that knockdown of PP2A Cα increases the expression of Osterix, leading to the accelerated osteoblast differentiation through the upregulation of bone-related genes. In this study, we examined whether Dlx5 and Runx2 are involved in the upregulated Osterix expression in PP2A Cα-knockdown osteoblasts (shPP2A cells). The expression of Dlx5 as well as Osterix was significantly higher in shPP2A cells in the initial stage of osteoblast differentiation compared with the control cells (shCont). The expression of Runx2 protein was also higher in shPP2A cells compared with shCont cells although its mRNA level was comparable. Reduction of Dlx5 or Runx2 decreased Osterix expression and alkaline phosphatase activity in shPP2A cells. Luciferase assay showed that Osterix promoter activity was drastically elevated in shPP2A cells compared with that in shCont cells. The deletion or mutation of the Dlx5 and Runx2 binding sites significantly suppressed Osterix promoter activity in shPP2A cells. These results indicate that Dlx5 and Runx2 are critical factors for the upregulated Osterix expression in shPP2A cells, which is considered to be important for the accelerated osteoblast differentiation in these cells.
丝氨酸/苏氨酸蛋白磷酸酶 2A（PP2A）参与调节多种生理过程，包括细胞周期、生长、凋亡和信号转导。成骨细胞分化受主要的骨特异性转录因子控制，包括成骨特异性转录因子 2（osterix）、远侧板状同源框 5（Dlx5）和 runt 相关转录因子 2（Runx2）。我们之前报道过，PP2A Cα 的敲低会增加 Osterix 的表达，通过上调与骨相关的基因，加速成骨细胞分化。在这项研究中，我们研究了 Dlx5 和 Runx2 是否参与了 PP2A Cα 敲低成骨细胞（shPP2A 细胞）中 Osterix 表达的上调。在成骨细胞分化的初始阶段，与对照细胞（shCont）相比，shPP2A 细胞中 Dlx5 和 Osterix 的表达明显更高。尽管 shPP2A 细胞中 Runx2 蛋白的表达也高于 shCont 细胞，但其 mRNA 水平相当。降低 Dlx5 或 Runx2 的表达会降低 shPP2A 细胞中 Osterix 的表达和碱性磷酸酶活性。荧光素酶检测显示，与 shCont 细胞相比，shPP2A 细胞中 Osterix 启动子活性显著升高。Dlx5 和 Runx2 结合位点的缺失或突变显著抑制了 shPP2A 细胞中 Osterix 启动子活性。这些结果表明，Dlx5 和 Runx2 是 shPP2A 细胞中 Osterix 表达上调的关键因素，这被认为对这些细胞中成骨细胞分化的加速很重要。
