Department of Biochemistry, School of Dentistry, IHBR, Kyungpook National University, Daegu, South Korea.
Department of Oral Medicine, School of Dentistry, IHBR, Kyungpook National University, Daegu, South Korea.
J Cell Physiol. 2023 Jul;238(7):1520-1529. doi: 10.1002/jcp.31024. Epub 2023 Apr 26.
To understand the mechanisms underlying tooth morphogenesis, we examined the developmental roles of important posttranslational modification, O-GlcNAcylation, which regulates protein stability and activity by the addition and removal of a single sugar (O-GlcNAc) to the serine or threonine residue of the intracellular proteins. Tissue and developmental stage-specific immunostaining results against O-GlcNAc and O-GlcNAc transferase (OGT) in developing tooth germs would suggest that O-GlcNAcylation is involved in tooth morphogenesis, particularly in the cap and secretory stage. To evaluate the developmental function of OGT-mediated O-GlcNAcylation, we employed an in vitro tooth germ culture method at E14.5, cap stage before secretory stage, for 1 and 2 days, with or without OSMI-1, a small molecule OGT inhibitor. To examine the mineralization levels and morphological changes, we performed renal capsule transplantation for one and three weeks after 2 days of in vitro culture at E14.5 with OSMI-1 treatment. After OGT inhibition, morphological and molecular alterations were examined using histology, immunohistochemistry, real-time quantitative polymerase chain reaction, in situ hybridization, scanning electron microscopy, and ground sectioning. Overall, inhibition of OGT resulted in altered cellular physiology, including proliferation, apoptosis, and epithelial rearrangements, with significant changes in the expression patterns of β-catenin, fibroblast growth factor 4 (fgf4), and sonic hedgehog (Shh). Moreover, renal capsule transplantation and immunolocalizations of Amelogenin and Nestin results revealed that OGT-inhibited tooth germs at cap stage exhibited with structural changes in cuspal morphogenesis, amelogenesis, and dentinogenesis of the mineralized tooth. Overall, we suggest that OGT-mediated O-GlcNAcylation regulates cell signaling and physiology in primary enamel knot during tooth development, thus playing an important role in mouse molar morphogenesis.
为了理解牙齿形态发生的机制，我们研究了重要的翻译后修饰 O-GlcNAcylation 的发育作用，该修饰通过在细胞内蛋白的丝氨酸或苏氨酸残基上添加和去除一个单糖（O-GlcNAc）来调节蛋白质的稳定性和活性。针对发育中牙胚中的 O-GlcNAc 和 O-GlcNAc 转移酶（OGT）的组织和发育阶段特异性免疫染色结果表明，O-GlcNAcylation 参与牙齿形态发生，特别是在帽状期和分泌期。为了评估 OGT 介导的 O-GlcNAcylation 的发育功能，我们在 E14.5 时采用了体外牙胚培养方法，即在分泌期之前的帽状期进行 1 天和 2 天的培养，有或没有 OSMI-1，一种小分子 OGT 抑制剂。为了检查矿化水平和形态变化，我们在 E14.5 时进行了体外培养 2 天后的肾囊移植，持续 1 周和 3 周，并进行了 OSMI-1 处理。在 OGT 抑制后，使用组织学、免疫组织化学、实时定量聚合酶链反应、原位杂交、扫描电子显微镜和地面切片检查形态和分子变化。总的来说，OGT 的抑制导致细胞生理发生改变，包括增殖、凋亡和上皮重排，β-catenin、成纤维细胞生长因子 4（fgf4）和 Sonic Hedgehog（Shh）的表达模式发生显著变化。此外，肾囊移植和 Amelogenin 和 Nestin 的免疫定位结果表明，在帽状期的 OGT 抑制的牙胚中，牙尖形态发生、釉质发生和矿化牙的牙本质发生表现出结构变化。总的来说，我们认为 OGT 介导的 O-GlcNAcylation 调节牙齿发育过程中初级釉结细胞中的细胞信号和生理学，因此在小鼠磨牙形态发生中发挥重要作用。
