The aim of this study was to evaluate the effect of melatonin on ameloblasts and to explore the signaling pathway of melatonin. The proliferation, differentiation, and mineralization of ameloblast-lineage cells, induced by melatonin, were studied using various techniques. The most widely used ameloblast-like cells are the ameloblast-like cells (LS-8) and the ameloblast-lineage cell line (ALC). By comparing the expression profiles of LS-8 cells and ALC cells, Sarkar et al. found that LS-8 cells expressed more genes representing secretory-stage activities (amelx, AMBN, ENAM, and MMP20), while ALC expressed more genes representing the maturation-stage activities (ODAM and KLK4). In addition, ALC cells in the basic medium could form calcified nodules, while LS-8 cells did not . To better understand the differentiation and mineralization of ameloblasts, ALC was selected.
At present, research on melatonin in oral hard tissue mainly focuses on promoting the growth and development of dentin and alveolar bone, as well as the induction and differentiation of dental pulp stem cells [30–32]. However, the effect of melatonin on ameloblasts remains unclear. In this study, we confirmed that a melatonin concentration of 10-3 M inhibited the proliferation of ALC cells in a time- and concentration-dependent manner. Melatonin has been shown to effectively modulate the proliferation of various types of cells . For example, 1 mM melatonin inhibits the proliferation of breast cancer MCF-7  and umbilical vein endothelial cells  in vitro. Melatonin (10-6–10-4 M) has no obvious effect on the proliferation of human osteoblast cell line hFOB1.19 , but melatonin (10-12–10-10 M) has an obvious inhibitory effect on the proliferation of human dental pulp cells or HDPCs . This evidence suggests that melatonin may have different sensitivity and specificity in inhibiting cell proliferation in vitro. In order to observe the effect of melatonin on ALC cells more intuitively, the morphology of ALC cells will be detected by laser scanning confocal microscopy. We showed that ALC cells were more evenly distributed and produced more pseudopodia in the melatonin medium than in the standard medium. To some extent, with the increase in melatonin concentration, myofilament proteins were more densely arranged and interwoven, but the number of cells had decreased at the same time, which was similar to the results of the cell proliferation experiment. The changes in cell number and myofilament protein levels were not obvious at 48 h, compared with 96 h. Therefore, we speculate that melatonin promotes actin fiber production and increases cytoskeleton construction in a time- and concentration-dependent manner.
Exogenous melatonin can restore and stabilize the circadian rhythm of mice with the pineal gland removed .In our previous work, the loss of circadian rhythm mainly inhibited enamel formation and AMELX expression in mice through the melatonin receptor . The role of ALC cells in the formation of mineralized tissues is similar to that of mouse ameloblasts. Therefore, we hypothesized that melatonin may promote the amelogenic behavior of ALC cells. According to the experimental results, melatonin receptors MT1 and MT2 are present and stably expressed in ALC cell lines, suggesting that ameloblasts may be one of the targets of melatonin. We found that the expression of amelogenes in ALC cells was significantly enhanced by 10-3 M melatonin. In the present study, we analyzed three important enamel matrix proteins, amelogenin X-linked (AMELX), enamelin (ENAM), and odontogenic ameloblast-associated protein (ODAM). AMELX is secreted by ameloblasts, accounting for approximately 95% of enamel matrix proteins. It is the most important matrix for enamel formation, maturation, and mineralization. It mainly regulates the crystallographic structure and thickness of the enamel . ENAM is an important component of non-amelogenin, which can promote crystal nucleation, and the crystal size increases with an increase in its concentration [40, 41]. ODAM is also produced by ameloblasts, at low levels. Although it is not clearly established, ODAM is considered one of the regulators of enamel mineralization and crystal elongation . The expression curve of AMELX mRNA was consistent with that of MT1 and MT2 mRNA, which peaked on the third day and then began to decline. This suggests that melatonin may promote the expression of AMELX through its receptors MT1 and MT2, thus stimulating the differentiation of enamel cells, which is consistent with previous experimental results in vivo. However, the expression curve of ENAM and ODAM was relatively stable within 5 days. There was a significant difference compared with the control group, and it did not change with the melatonin receptor expression. This suggests that melatonin also promotes the expression of ENAM and ODAM, but it may be through a receptor-independent pathway.
In addition to regulating the biomolecules of the enamel organic matrix, melatonin can further manipulate the amelogenin behaviors of ameloblasts. High concentrations of melatonin, especially 10-3 M, improved ameloblast functions, as confirmed by microscopic observations of ALP synthesis and mineralized nodule formation. According to the observed ALP activity levels, melatonin can promote ALP activity in ALC cells in a concentration-dependent manner. According to the enamel formation patterns observed at different concentrations of melatonin, the number of calcified nodules and the positive staining degree of Alizarin Red increased with increasing melatonin concentration. As ALC cell lines have the ability to form mineralization, we speculate that melatonin mainly promotes the formation of calcified nodules in ameloblasts rather than by induction. At the same time, luzindole, a melatonin receptor inhibitor, significantly inhibited the calcification of ALC, which suggested that melatonin could regulate the calcification of ameloblasts through MT1 and MT2.
According to the above conclusions, it can be concluded that melatonin inhibits proliferation and promotes the differentiation of ALC cells. Therefore, it is particularly important to further explore the specific molecular mechanisms of melatonin in the growth and development of tooth enamel cells . Studies have shown that Wnt/β-catenin signaling pathways are critical for bone and tooth formation and development, and are involved in various stages of tooth morphogenesis, such as epithelial-mesenchymal interaction, tooth-specific cell proliferation, migration, and differentiation . Signaling factors in various Wnt pathways are expressed in the inner enamel epithelium and ameloblast cells . Activation of Wnt/β-catenin signaling may stimulate osteoblast differentiation and osteogenesis . The inactivation of β-catenin during the development of odontoblast cells can lead to a deficiency in the roots of the molar [45, 46].
In this study, RNA-seq data showed that the genes associated with the Wnt pathway changed significantly, indicating that the activity of Wnt signaling pathway was significantly changed during melatonin treatment of ALCS. Interestingly, the expression of the FZD receptor was significantly increased, and the WNT ligand was generally decreased. This finding was consistent with the results of qRT-PCR, in which Wnt4 and Wnt6 were 7- and 11-fold downregulated, respectively. Studies have shown that members of the Wnt5a family, including Wnt4, Wnt5a, and Wnt11, not only effectively activate the canonical pathway, but also inhibit it(Maye, Zheng et al. 2004). Therefore, we speculated that melatonin activates the canonical pathway by inhibiting the expression of Wnt5a family factors. We then used qRT-PCR to detect the significant signaling molecules involved in the Wnt/β-catenin signaling pathway. Among them, the levels of β-catenin and LEF-1 were significantly upregulated to activate downstream factors. The expression of c-Jun decreased, and that of Cyclin D1 did not change significantly. These findings indicated that melatonin may pass through Wnt/ β-Catenin pathway to play a role in ALCs. However, there are many signaling pathways of melatonin in enamel development, which need to be further explored and verified. At the same time, cell lines are unable to simulate the environment in vivo, so future studies will focus on the validation of these hypotheses using vivo and animal models for pre-clinical trials.