Abstract
Introduction: 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. Objective: 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. Method: 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. Results: 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.
Highlights
Melatonin, an endogenous neurohormone, modulates the biological circadian rhythms of vertebrates
In the area of molecular developing biology, melatonin is referred to tooth development, by regulating cellular processes in odontogenic cells (Permuy et al, 2017), altering mitochondrial activity in dental papilla cells (Liu et al, 2013; Jiang et al, 2019), et al In previous study, we have discovered the gene and protein expression of melatonin receptors (MTNR) in ALCs (Supplementary Figure S1C,D)
What’s more, mounting evidence in this study indicated that melatonin does promote the mineralization of ALCs through melatonin receptor dependent pathway in vitro, for instance, stimulating the expression of enamel matrix proteins (EMPs) and the activity of ALP, promoting the deposition of calcified nodules
Summary
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. Mature enamel is the hardest, most mineralized tissue in the human body, comprising >95% by weight crystals of substituted calcium hydroxyapatite (HA; Ca10 [PO4]6 [OH]2), which is secreted by the ameloblasts, the enamel-forming cells (Lacruz et al, 2017). Enamel lacks any capacity for cellular repair and once formed, must function over a lifetime (Smith et al, 2017). In view of the non-renewable nature of enamel, it is urgent and necessary for us to study the mechanism of amelogenesis imperfect
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