Zheng H, Fu J, Chen Z, Yang G, Yuan G
The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China.
Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China.
J Dent Res. 2022 Aug;101(9):1064-1074. doi: 10.1177/00220345221077202. Epub 2022 Feb 26.
Dentin is a major mineralized component of teeth. Odontoblasts are responsible for synthesis and secretion of dentin matrix. Previously, it has been demonstrated in a cell culture system that the E3 ubiquitin ligase, murine double minute 2 (Mdm2), promotes odontoblast-like differentiation of mouse dental papilla cells (mDPCs) by ubiquitinating p53 and the odontoblast-specific substrate Dlx3. However, whether Mdm2 plays an essential role in vivo in odontoblast differentiation and dentin formation remains unknown. In this study, we investigated the in vivo functions of Mdm2 using mice combined with multiple histological and molecular biological methods. The results showed that Mdm2 deletion in the odontoblast layer led to defects in odontoblast differentiation and dentin formation. Unexpectedly, specific inhibition of the Mdm2-p53 axis in wild-type mice by injection of a small-molecule inhibitor Nutlin-3a indicated that the role of Mdm2 in dentinogenesis was p53 independent, which was inconsistent with the previous in vitro study. In situ proximity ligation assay (PLA) showed that Mdm2 interacted with and ubiquitinated Dlx3 in the odontoblast nucleus of mouse molars. Dlx3 promoted the translocation of Mdm2 to the nucleus, and in turn, the nuclear Mdm2 mediated ubiquitination of Dlx3 and promoted the odontoblast-like differentiation of mDPCs. Dlx3 interacted with Mdm2 through its C-terminal domain. Deletion of the C-terminal domain of Dlx3 reversed the enhanced odontoblast-like differentiation and the activation of promoter mediated by overexpression of wild-type or nuclear Mdm2. Our findings suggest that nuclear Mdm2 mediates ubiquitination of the transcription factor Dlx3, which is essential for Dlx3 transcriptional activity on as well as subsequent odontoblast differentiation and dentin formation.
牙本质是牙齿的主要矿化成分。成牙本质细胞负责牙本质基质的合成与分泌。此前，在细胞培养系统中已证实，E3泛素连接酶小鼠双微体2（Mdm2）通过使p53和牙本质特异性底物Dlx3泛素化，促进小鼠牙乳头细胞（mDPCs）向成牙本质样细胞分化。然而，Mdm2在成牙本质细胞分化和牙本质形成的体内过程中是否发挥关键作用仍不清楚。在本研究中，我们结合多种组织学和分子生物学方法，利用小鼠研究了Mdm2的体内功能。结果表明，成牙本质细胞层中Mdm2的缺失导致成牙本质细胞分化和牙本质形成缺陷。出乎意料的是，通过注射小分子抑制剂Nutlin-3a对野生型小鼠的Mdm2-p53轴进行特异性抑制表明，Mdm2在牙本质形成中的作用不依赖于p53，这与之前的体外研究结果不一致。原位邻近连接分析（PLA）显示，Mdm2在小鼠磨牙的成牙本质细胞核中与Dlx3相互作用并使其泛素化。Dlx3促进Mdm2向细胞核的转位，反过来，细胞核中的Mdm2介导Dlx3的泛素化，并促进mDPCs向成牙本质样细胞分化。Dlx3通过其C末端结构域与Mdm2相互作用。缺失Dlx3的C末端结构域可逆转由野生型或细胞核Mdm2过表达介导的成牙本质样细胞分化增强和启动子激活。我们的研究结果表明，细胞核中的Mdm2介导转录因子Dlx3的泛素化，这对于Dlx3对[具体基因]的转录活性以及随后的成牙本质细胞分化和牙本质形成至关重要。
