Circadian rhythms regulate amelogenesis

Abstract

Ameloblasts, the cells responsible for making enamel, modify their morphological features in response to specialized functions necessary for synchronized ameloblast differentiation and enamel formation. Secretory and maturation ameloblasts are characterized by the expression of stage-specific genes which follows strictly controlled repetitive patterns. Circadian rhythms are recognized as key regulators of the development and diseases of many tissues including bone. Our aim was to gain novel insights on the role of clock genes in enamel formation and to explore the potential links between circadian rhythms and amelogenesis. Our data shows definitive evidence that the main clock genes (Bmal1, Clock, Per1 and Per2) oscillate in ameloblasts at regular circadian (24 h) intervals both at RNA and protein levels. This study also reveals that the two markers of ameloblast differentiation i.e. amelogenin (Amelx; a marker of secretory stage ameloblasts) and kallikrein-related peptidase 4 (Klk4, a marker of maturation stage ameloblasts) are downstream targets of clock genes. Both, Amelx and Klk4 show 24h oscillatory expression patterns and their expression levels are up-regulated after Bmal1 over-expression in HAT-7 ameloblast cells. Taken together, these data suggest that both the secretory and the maturation stages of amelogenesis might be under circadian control. Changes in clock gene expression patterns might result in significant alterations of enamel apposition and mineralization.

Fig. 1

Clock gene RNAs follow circadian rhythms in HAT-7 cell line. A) Bmal1 RNA highest expression levels are observed at ZT20. B) Clock RNA highest expression levels are found at ZT 20. C) Per1 RNA shows highest expression levels at ZT12. D) Per2 shows the strongest expression levels at ZT10-ZT12. Data are means ± S.D. from at least three independent replicates.

Fig. 2

Prediction of expression rhythm phase by Fast Fourier analysis is done using time series data of Bmla1 and Per2. 10000 random permutations of the time series data are used to calculate the probability of obtaining the power value by chance (significance level of 1%). The p-value is shown in the figure.

Fig. 3

Clock gene proteins exhibit circadian rhythms in HAT-7 cell line. A) Western blot data demonstrate that the expression levels of clock proteins BMAL1, CLOCK, PER1, PER2 and of two ameloblast-specific proteins, AMELX and KLK4, follow circadian rhythms. After analyzed using ImageJ software, results show that B) BMAL1 protein expression levels arrive to zenith at ZT4; C) CLOCK protein expression reaches the highest levels at ZT0; D) PER1 proteins show strong expression levels at ZT12; E) Both PER2 detected protein isoforms exhibit the highest expression peak at ZT8 after synchronization. Data are presented as means± S.D. from three independent replicates.

Fig. 4

Cellular localization of clock proteins in HAT-7 during a 24h period. A) Immunocytofluorescence staining reveals that BMAL1 proteins are mostly localized in the cytoplasm at ZT0 and then shift into the nucleus at ZT12 (left). In contrast, PER2 is localized in the cell nucleus at ZT0 and then it shifts to the cytoplasm at ZT12 (right). B) IHC results show that BMAL1 (C) and PER2 (D) are expressed in ameloblasts (black arrows). On serial sections, BMAL1 is mostly expressed in the cytoplasm (D), and PER2 is localized mainly in the nucleus (E). Most of BMAL1 proteins are localized in the cytoplasm at 8 am and then in the nucleus of ameloblasts at 8 pm (F). PER2 proteins are found in the nucleus at 8 am and in the cytoplasm of ameloblasts at 8 pm. Am, ameloblast; od, odontoblast; nu, nucleus; cp, cytoplasm. Scale bars = 20 μm in A, 200 μm in B and C, 40 μm in D and E, 10 μm in F and G.

Fig. 5

Ameloblast-specific genes expression exhibits circadian rhythms in HAT-7. A) Real-time qRT-PCR reveals that Amelx, a marker of secretory amelobalsts, RNA expression follows a circadian pattern in HAT-7 cells after cell cycle synchronization. B) Klk4, a marker of maturation ameloblsts, also exhibits RNA circadian pattern of expression. At the protein levels, after ImageJ analysis, AMELX shows the highest protein expression levels at ZT0 (C), and KLK4 protein shows the highest level of expression at ZT4 (D). Klk4 relative expression levels are slightly higher than Amelx relative expression levels in HAT-7 cells but both genes follow clear circadian patterns. Over-expression of Bmal1 cDNA results in up-regulation of Amexl and Klk4 RNAs expression in HAT-7 (E). In contrast, Enamelin (which is another ameloblast-specific gene) RNA expression remains unchanged. Over-expression of Cry1 (F) results in down-regulation of Bmal1 (G). Amelx and Klk4 expression levels also are significantly down-regulated (H). The representative data are presented as means ± S.D. from at least three independent replicates where data from cells transfected with Bmla1 or Cry1 were compared with cells transfected with empty vector. Statistical significance (*) was considered when p<0.05.