O‐GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics‐oriented study
O‐GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics‐oriented study
复制标题
DOI：
10.1002/biof.1774
复制
发表时间：
2021-08
期刊：
影响因子：
6
通讯作者：
Yao Weng;Ziyi Wang;Y. Fukuhara;A. Tanai;M. Ikegame;D. Yamada;T. Takarada;T. Izawa;S. Hayano;Kaya Yoshida;H. Kamioka;H. Okamura
中科院分区：
文献类型：
--
作者：
Yao Weng;Ziyi Wang;Y. Fukuhara;A. Tanai;M. Ikegame;D. Yamada;T. Takarada;T. Izawa;S. Hayano;Kaya Yoshida;H. Kamioka;H. Okamura
本研究旨在通过一系列以生物信息学为导向的实验，揭示O-连接-N-乙酰氨基葡萄糖化（O-GlcNAc化）调控成骨细胞分化的可能机制。为了检查O-GlcNAc化水平对成骨细胞分化的影响，用O-GlcNAc转移酶（OGT）和O-GlcNAc酶（OGA）抑制剂处理成骨细胞MC 3 T3-E1细胞。通过qPCR和蛋白质印迹法评价O-GlcNAc化水平与成骨标志物表达以及OGT之间的相关性。从几个公共数据库中检索假定与Runx 2表达相关的O-GlcNAc化蛋白质，并用于进一步的生物信息学分析。根据生物信息学分析的结果，使用共聚焦激光扫描显微镜（CLS）监测用OGT和OGA抑制剂处理的细胞中的细胞内钙（[Ca 2 +]i）。使用稳定的OGT敲低MC 3 T3-E1细胞测定O-GlcNAc化和[Ca 2 +]i对成骨标志物表达的相互作用。O-GlcNAc化与成骨细胞分化呈正相关。在成骨细胞分化过程中，整体O-GlcNAc酰化蛋白的时程曲线显示出不同分子量的独特模式。几种O-GlcNAc酰化蛋白的表达模式与Runx 2表达的表达模式显著相似。对检索到的Runx 2相关-O-GlcNAcylated-蛋白的生物信息学分析揭示了[Ca 2 +]i的重要性。CLS显示O-GlcNAc化的改变迅速改变MC 3 T3-E1细胞中的[Ca 2 +]i。O-GlcNAc化和[Ca 2 +]i对成骨标志物的表达表现出交互作用。OGT敲除破坏了[Ca 2 +]i诱导的成骨标志物表达变化。O-GlcNAc化与[Ca 2 +]i相互作用，通过调节成骨标志物的表达促进成骨细胞分化。
This study aimed to reveal the possible mechanisms by which O‐linked‐N‐acetylglucosaminylation (O‐GlcNAcylation) regulates osteoblast differentiation using a series of bioinformatics‐oriented experiments. To examine the influence of O‐GlcNAcylation levels on osteoblast differentiation, osteoblastic MC3T3‐E1 cells were treated with O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) inhibitors. Correlations between the levels of O‐GlcNAcylation and the expression of osteogenic markers as well as OGT were evaluated by qPCR and western blotting. The O‐GlcNAcylated proteins assumed to correlate with Runx2 expression were retrieved from several public databases and used for further bioinformatics analysis. Following the findings of the bioinformatics analysis, intracellular calcium ([Ca2+]i) was monitored in the cells treated with OGT and OGA inhibitors using a confocal laser‐scanning microscope (CLS). The interaction effect between O‐GlcNAcylation and [Ca2+]i on osteogenic marker expression was determined using stable OGT knockdown MC3T3‐E1 cells. O‐GlcNAcylation was positively associated with osteoblast differentiation. The time‐course profile of global O‐GlcNAcylated proteins showed a distinctive pattern with different molecular weights during osteoblast differentiation. The expression pattern of several O‐GlcNAcylated proteins was significantly similar to that of Runx2 expression. Bioinformatic analysis of the retrieved Runx2‐related‐O‐GlcNAcylated‐proteins revealed the importance of [Ca2+]i. CLS showed that alteration of O‐GlcNAcylation rapidly changed [Ca2+]i in MC3T3‐E1 cells. O‐GlcNAcylation and [Ca2+]i showed an interaction effect on the expression of osteogenic markers. OGT knockdown disrupted the [Ca2+]i‐induced expression changes of osteogenic markers. O‐GlcNAcylation interacts with [Ca2+]i and elicits osteoblast differentiation by regulating the expression of osteogenic markers.