Abstract
Circadian rhythm is involved in the development and diseases of many tissues. However, as an essential environmental regulating factor, its effect on amelogenesis has not been fully elucidated. The present study aims to investigate the correlation between circadian rhythm and ameloblast differentiation and to explore the mechanism by which circadian genes regulate ameloblast differentiation. Circadian disruption models were constructed in mice for in vivo experiments. An ameloblast-lineage cell (ALC) line was used for in vitro studies. As essential molecules of the circadian system, Bmal1 and Per2 exhibited circadian expression in ALCs. Circadian disruption mice showed reduced amelogenin (AMELX) expression and enamel matrix secretion and downregulated expression of BMAL1, PER2, PPARγ, phosphorylated AKT1 and β-catenin, cytokeratin-14 and F-actin in ameloblasts. According to previous findings and our study, BMAL1 positively regulated PER2. Therefore, the present study focused on PER2-mediated ameloblast differentiation and enamel formation. Per2 knockdown decreased the expression of AMELX, PPARγ, phosphorylated AKT1 and β-catenin, promoted nuclear β-catenin accumulation, inhibited mineralization and altered the subcellular localization of E-cadherin in ALCs. Overexpression of PPARγ partially reversed the above results in Per2-knockdown ALCs. Furthermore, in in vivo experiments, the length of incisor eruption was significantly decreased in the circadian disturbance group compared to that in the control group, which was rescued by using a PPARγ agonist in circadian disturbance mice. In conclusion, through regulation of the PPARγ/AKT1/β-catenin signalling axis, PER2 played roles in amelogenin expression, cell junctions and arrangement, enamel matrix secretion and mineralization during ameloblast differentiation, which exert effects on enamel formation.
Highlights
Circadian rhythm is involved in most physiological processes.[1]
We found that circadian disruption in mice resulted in molecular dysregulation, including decreased BMAL1 and PER2 and reduced enamel matrix formation during amelogenesis
Bmal1-knockdown ameloblast-lineage cell (ALC) cells showed decreased protein expression of PER2 and PPARγ, while the expression of BMAL1 presented no significant variation in ALC-Per2sh cells
Summary
The circadian rhythm coordinates cellular and organismal metabolism, including regulation of heart rate, growth hormone secretion, bone modelling, lipid homeostasis and so on.[4,5] Circadian gene products were detected during tooth development in mice, which indicated that circadian rhythm could play roles in odontogenesis.[16]. Previous studies demonstrated that PER2 is dampened when the circadian rhythm is disrupted.[31,32] Here, in circadian disruption mice, we observed decreased expression of PER2, PPARγ, AKT1Ser[473], β-catenin-Ser[552] and AMELX in ameloblasts, reduced enamel matrix secretion and decreased incisor eruption, showing the possible association between these molecules and circadian disturbed ameloblast differentiation. The study presented findings showing that circadian disruption in mice resulted in reduced enamel matrix formation and molecular dysregulation during amelogenesis It Relative protein expression Relative protein expression. *P < 0.05; **P < 0.01 was further revealed that PER2, an essential molecule of the circadian system, exerted pivotal effects on the function and differentiation of ameloblasts, including amelogenin expression, cell junctions, enamel matrix secretion and mineralization, which were regulated by the PPARγ/AKT1/β-catenin signalling axis. The findings provide insights to understand the association and mechanisms between circadian rhythm and amelogenesis and propose a new perspective for the prevention of developmental defects of enamel
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