Elsevier

Bone

Volume 55, Issue 1, July 2013, Pages 158-165
Bone

Original Full Length Article
Circadian rhythms regulate amelogenesis

https://doi.org/10.1016/j.bone.2013.02.011Get rights and content

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 24 h 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.

Highlights

► Several clock proteins show circadian oscillatory expression patterns in ameloblast cells. ► BMAL1 and PER2 protein localization shifts from the nucleus to the cytoplasm in regular 12 h intervals in ameloblasts. ► CRY1 and BMAL1 proteins regulate Amelx and Klk4 RNA and protein levels in ameloblasts. ► Our data suggest that amelogenesis might be under circadian control.

Introduction

Circadian rhythms are self-sustained endogenous oscillations occurring over a 24 hour (h) period. These biological rhythms are involved in many physiological processes. Though there is a site in the suprachiasmatic nucleus of the brain that is considered the “master clock”, “peripheral clocks” have been found in several tissues in the body. Peripherals clocks may respond to the environmental light–dark cycles of an organism but can persist oscillating even after the light–dark stimulus is removed. The relationship between the “master clock” and the “peripheral clocks” has not been made clear [1], [2].

Several genes, named “clock genes” have been identified as core maintainers of circadian rhythms. The main mammalian clock genes include Circadian Locomotor Output Cycles Kaput (Clock), Brain and Muscle Aryl Hydrocarbon Receptor Nuclear Translocation (ARNT)-like (Bmal1), Period 1 (Per1), Period 2 (Per2), Period 3 (Per3), and Cryptochromes (Cry1) and Cry2. Additionally, nuclear receptor subfamily 1, group D, members 1 and 2 (Nr1d1 and Nr1d2), RAR-Related Orphan Receptor Alpha (Rora), and Albumin d-binding protein (Dbp) also play a key role in circadian rhythm biology by regulating the expression of the main mammalian clock genes [3]. Transcription of the clock genes oscillates over an approximate 24 h period and their output signals induce rhythms of gene expression that create repetitive patterns in physiological processes. This method of circadian control involves binding of clock genes to the promoter region of clock-controlled genes (CCG) [4]. Binding to the promoter of target genes (CCG) occurs via three specific DNA sequences called E-, D-, and RRE-box [3]. In a more indirect way, clock genes may also bind to intermediate clock-controlled genes, such as key transcription factors, which then influence the expression of downstream target genes involved in cell proliferation or cell differentiation processes [1].

Clock genes regulate circadian rhythms and metabolic functions in mammals but have also been implicated in mineralized tissue development, including bone formation [5]. More recent studies have indicated that Cry2 and Per2 affect distinct pathways in the regulation of bone volume. More specifically, Cry2 influences mostly the osteoclastic cellular component of bone and Per2 acts on osteoblast's parameters [6].

Similar to bone, teeth are formed incrementally and previous studies suggest the existence of molecular clocks in enamel forming ameloblasts [7], [8] and in dentin forming odontoblasts [9]. Indeed, several lines of evidence support the idea that ameloblasts and odontoblasts are under circadian control. First, there are daily growth patterns found in enamel and dentin that are produced during the secretory stage and seem to follow circadian rhythms. These daily growth lines, called cross-striations in enamel, mark the amount of matrix deposited each 24 h by the ameloblast cells [10]. Second, it has been demonstrated that odontoblasts show circadian rhythms with regard to collagen synthesis and secretion [9]. It has also been suggested that these rhythms may be responsible for the circadian incremental lines observed in dentin [9]. Third, in addition to the circadian matrix deposition during secretory stage, ameloblasts alternate between two functionally and morphologically distinct cell forms every 8 h in rats during maturation stage i.e. the smooth- and ruffle-ended ameloblasts [11]. Ruffle-ended ameloblasts transport calcium and phosphate ions in the enamel matrix, whereas when smooth-ended ameloblast degrade proteins of the matrix [11], [12]. Although the above observations strongly suggest that dental tissue formation is under circadian control, no clear evidence for a “dental” circadian clock exists. It is also still unclear how circadian control affects ameloblast and odontoblast functions and dental tissue formation resulting in fully mineralized enamel and dentin.

We recently reported evidence that clock genes are differentially expressed during tooth development, mainly by ameloblasts and odontoblasts [8], [13]. Furthermore, we provided preliminary evidence that the clock gene Nr1d1 is expressed and oscillates in 24 h intervals in an ameloblast cell line (HAT-7) [14]. We also found that key ameloblast markers such as amelogenin may be under the control of Nr1d1 [14]. Consistently, we have showed that the total amount of enamel secreted proteins follows daily biological rhythms [8]. However, a detailed analysis of clock gene circadian expression in ameloblasts and the evaluation of their multi-level control in ameloblast genes is still missing. This research was undertaken to increase our knowledge on the role of clock genes in enamel formation.

Section snippets

Cell culture, synchronization and transfection studies

HAT-7 [15] cells are maintained in DMEM/F-12 (Sigma, St. Louis, MO) supplemented with 100 units/ml penicillin G, 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA) and 10% (v/v) FBS (fetal bovine serum; SAFC Biosciences, Lenexa, KS). To induce cell cycle synchronization, cells are exposed for 2 h to serum-free medium containing 15 μM forskolin (FSK; Calbiochem, La Jolla, CA), as previously described [16]. After that, cells are changed to regular culture medium. All time interval calculations are

Clock RNAs follow circadian rhythms in HAT-7 cell line

We first used qRT-PCR to evaluate if clock gene RNAs exhibit a circadian rhythm in HAT-7 cells, an ameloblast cell line, after cell cycle synchronization. Circadian rhythms are evaluated usually in 6 consecutive 4-hour intervals (full 24 h period) as previously described [17]. We extended our analysis to two consecutive 24 h periods as an additional confirmation of the existence of circadian rhythms in ameloblasts. Rhythmic expression patterns are found for two consecutive days for all clock RNAs

Discussion

Although previous research from our group [8], [13], [14] and others [7], [18] has suggested that enamel formation might be controlled by circadian rhythms little is known regarding the mechanisms of clock gene control in amelogenesis. Here, we conducted a series of studies to clarify how clock genes and their products may guide ameloblast function by controlling ameloblast-specific genes at RNA and protein expression levels. Our data supports a key role of clock genes in ameloblast functions.

Acknowledgments

This research was funded by the funds provided by the Department of Orthodontics and Pediatric Dentistry, School of Dentistry, University of Michigan and NIH grant DE018878 to PP.

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