Ren Qianhui, Pan Jing, Chen Yunshuo, Shen Zhecheng, Yang Zhao, Kwon Kubin, Guo Ying, Wang Yueying, Ji Fang
Shanghai Key Laboratory of Stomatology, National Center for Stomatology, National Clinical Research Center for Oral Diseases, Department of Orthodontics, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, College of Stomatology, Shanghai Jiao Tong University, Shanghai, China.
State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Rui Jin Hospital, Shanghai Institute of Hematology, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Front Cell Dev Biol. 2021 Dec 24;9:749642. doi: 10.3389/fcell.2021.749642. eCollection 2021.
Melatonin, an endogenous neurohormone, modulates the biological circadian rhythms of vertebrates. It functions have been reported in previous stomatological studies as anti-inflammation, antioxidant, osseointegration of dental implants and stimulation to dental pulp stem cells differentiation, but its role in ameloblastic differentiation and mineralization has been rarely studied. To reveal the effects of melatonin on the mineralization of ameloblast lineage cells (ALCs), and to identify the change in gene expression and the potential mechanism based on ribonucleic acid sequencing (RNA-seq) analysis. ALCs were induced in melatonin-conditioned medium. After 7-days culture, Western blot, real-time PCR, alkaline phosphatase (ALP) activity test, RNA-seq were accordingly used to detect the change in molecular level. After 1-month odontogenic induction in melatonin medium, Alizarin Red-S (ARS) staining showed the changes of mineral nodules. Differentially expressed genes (DEGs), enrichment of functions and signaling pathways analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database were performed. The JNK3 antagonist (JNK3 inhibitor IX, SR3576) and β-arrestin1 (Arrb1) overexpression were applied to confirm the fluctuation of melatonin-medicated JNK3 and Arrb1 expression. In this study, we found out melatonin contributed to the ameloblastic mineralization, from which we can observed the elevated expression of enamel matrix protein, and increased ALP activity and mineralized nodules formation. RNA-seq analysis showed the up-regulation of neural JNK3 and down-regulation of Arrb1 in ALCs. Meanwhile, phosphorylated JNK3 deficiency (phosphorylated JNK3 inhibitor---SR3576 added to culture medium) led to mineralization delay, and Arrb1 overexpression proved Arrb1 takes bridge between melatonin receptors (MTNR) and JNK3 in MAPK signaling pathway.
褪黑素是一种内源性神经激素，可调节脊椎动物的生物昼夜节律。在以往的口腔医学研究中，已报道其具有抗炎、抗氧化、促进牙种植体骨整合以及刺激牙髓干细胞分化等功能，但其在成釉细胞分化和矿化中的作用鲜有研究。为揭示褪黑素对成釉细胞系细胞（ALCs）矿化的影响，并基于核糖核酸测序（RNA-seq）分析确定基因表达变化及潜在机制。将ALCs在褪黑素条件培养基中诱导培养。培养7天后，相应地采用蛋白质免疫印迹法、实时定量聚合酶链反应、碱性磷酸酶（ALP）活性检测、RNA-seq检测分子水平的变化。在褪黑素培养基中进行1个月的成牙诱导后，茜素红S（ARS）染色显示矿化结节的变化。基于京都基因与基因组百科全书（KEGG）和基因本体论（GO）数据库进行差异表达基因（DEGs）分析、功能富集和信号通路分析。应用JNK3拮抗剂（JNK3抑制剂IX，SR3576）和β-抑制蛋白1（Arrb1）过表达来证实褪黑素介导的JNK3和Arrb1表达的波动。在本研究中，我们发现褪黑素有助于成釉细胞矿化，从中可以观察到釉基质蛋白表达升高、ALP活性增加以及矿化结节形成。RNA-seq分析显示ALCs中神经JNK3上调而Arrb1下调。同时，磷酸化JNK3缺陷（向培养基中添加磷酸化JNK3抑制剂 - SR3576）导致矿化延迟，Arrb1过表达证明Arrb1在丝裂原活化蛋白激酶（MAPK）信号通路中在褪黑素受体（MTNR）和JNK3之间起桥梁作用。
