O-GlcNAc Modification of the runt-Related Transcription Factor 2 (Runx2) Links Osteogenesis and Nutrient Metabolism in Bone Marrow Mesenchymal Stem Cells
O-GlcNAc Modification of the runt-Related Transcription Factor 2 (Runx2) Links Osteogenesis and Nutrient Metabolism in Bone Marrow Mesenchymal Stem Cells
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DOI：
10.1074/mcp.m114.040691
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发表时间：
2014-12-01
影响因子：
7
通讯作者：
Ball, Lauren E.
中科院分区：
文献类型：
--
作者：
Nagel, Alexis K.;Ball, Lauren E.
Runx2是控制成骨细胞分化和矿化骨架形成的主开关。通过磷酸化、泛素化和乙酰化对Runx2进行翻译后修饰，可调节其活性、稳定性以及与成骨信号下游转录共调节因子和染色质重塑蛋白的相互作用。通过电子转移解离串联质谱对Runx2进行表征，揭示了O-linked N-acetylglucosamine （O-GlcNAc）修饰位点，这是一种营养反应性的翻译后修饰，可调节多种转录效应物的作用。O-GlcNAc修饰发生在磷酸化残基和已知调节Runx2转激活区域内精氨酸甲基化的新位点附近。还检测到Runx2与O-GlcNAc酰化的O-GlcNAc转移酶之间的相互作用。药理抑制O-GlcNAcase (OGA)，负责从Ser/Thr残基中去除O-GlcNAc的酶，增强MC3T3-E1前成骨细胞中基础（39.9%）和bmp2 /7诱导的Runx2转录活性（43.3%）。在成骨培养基中分化6天的骨髓间充质干细胞中，OGA的抑制导致碱性磷酸酶（ALP）的表达（24.3%）和活性（65.8%）升高，碱性磷酸酶是骨形成的早期标志物，也是Runx2的转录靶点。与未受刺激的细胞相比，骨髓间充质干细胞在BMP2/7存在下8天的成骨分化最终导致OGA活性下降（39.0%）和o - glcn酰化Runx2丰度增加。此外，在OGA抑制剂存在下分化的骨髓间充质干细胞中，bmp2 /7诱导的ALP活性增加了35.6%，这表明直接或bmp2 /7诱导的OGA抑制与ALP活性增加有关。总之，这些发现将O-GlcNAc循环与成骨细胞分化条件下早期ALP标志物的runx2依赖性调节联系起来。
Runx2 is the master switch controlling osteoblast differentiation and formation of the mineralized skeleton. The post-translational modification of Runx2 by phosphorylation, ubiquitinylation, and acetylation modulates its activity, stability, and interactions with transcriptional co-regulators and chromatin remodeling proteins downstream of osteogenic signals. Characterization of Runx2 by electron transfer dissociation tandem mass spectrometry revealed sites of O-linked N-acetylglucosamine (O-GlcNAc) modification, a nutrient-responsive post-translational modification that modulates the action of numerous transcriptional effectors. O-GlcNAc modification occurs in close proximity to phosphorylated residues and novel sites of arginine methylation within regions known to regulate Runx2 transactivation. An interaction between Runx2 and the O-GlcNAcylated, O-GlcNAc transferase enzyme was also detected. Pharmacological inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc from Ser/Thr residues, enhanced basal (39.9%) and BMP2/7-induced (43.3%) Runx2 transcriptional activity in MC3T3-E1 pre-osteoblasts. In bone marrow-derived mesenchymal stem cells differentiated for 6 days in osteogenic media, inhibition of OGA resulted in elevated expression (24.3%) and activity (65.8%) of alkaline phosphatase (ALP) an early marker of bone formation and a transcriptional target of Runx2. Osteogenic differentiation of bone marrow-derived mesenchymal stem cells in the presence of BMP2/7 for 8 days culminated in decreased OGA activity (39.0%) and an increase in the abundance of O-GlcNAcylated Runx2, as compared with unstimulated cells. Furthermore, BMP2/7-induced ALP activity was enhanced by 35.6% in bone marrow-derived mesenchymal stem cells differentiated in the presence of the OGA inhibitor, demonstrating that direct or BMP2/7induced inhibition of OGA is associated with increased ALP activity. Altogether, these findings link O-GlcNAc cycling to the Runx2-dependent regulation of the early ALP marker under osteoblast differentiation conditions.