Abstract
Core circadian clock genes set the pace for a wide range of physiological functions, including regeneration. The role of these genes and their regulation in the dental pulp, in particular under hypoxic conditions, is unknown. Here we investigated if core clock genes are expressed in human dental pulp‐derived cells (DPC) and if their expression is modulated by the hypoxia mimetic agent, L‐mimosine (L‐MIM), hypoxia or echinomycin. Dental pulp‐derived cells in monolayers and spheroids were treated with L‐MIM, hypoxia or echinomycin. mRNA levels of the core circadian clock genes were analysed using quantitative PCR (qPCR) and their protein levels were analysed by western blot. All core clock genes and proteins were produced in DPC monolayer and spheroid cultures. The expression of cryptochrome circadian regulators and period circadian regulators was reduced by L‐MIM, hypoxia and echinomycin at mRNA, but not at protein levels. Time course experiments indicated that modulations were based on alterations in overall mRNA levels of core circadian clock genes. Our results suggest a potential role of the core circadian clock in the response of dental pulp to hypoxia. Future studies need to consider that regulation of the core circadian clock at mRNA levels might not be paralleled by modulation of protein levels.
Highlights
The level of mRNA expressed by these clock genes relative to that of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was as follows: circadian locomotor output cycles kaput (CLOCK) (0.0021 Æ 0.0014), BMAL1 (0.0002 Æ 0.0002), CRY1 (0.0018 Æ 0.0026), CRY2 (0.0060 Æ 0.0079), period circadian regulator 1 (PER1) (0.0070 Æ 0.0094), PER2 (0.0012 Æ 0.0015) and PER3 (0.0033 Æ 0.0040)
The circadian clock has been proposed to be involved in tooth development, little is known about the role of core circadian clock genes and their regulation in the dental pulp [5]
There is a need for studies that aim to reveal the relevance of molecular clocks in the dental pulp
Summary
Organisms are able to synchronize a variety of molecular and physiological functions, such as behaviour, reproduction or cell division, with steady cycles of stimuli that come from the environment. The proteins circadian locomotor output cycles kaput (CLOCK) and aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) form a heterodimer They act as transcription factors when binding to the E-boxes in the promotor regions of period circadian regulator (PER1, PER2, PER3) and cryptochrome circadian regulator (CRY1, CRY2) genes. A role of molecular clocks has been proposed in oral tissue and tooth development, the existence and function of the core clock genes in the dental pulp is currently unclear [6]. Understanding the principles of molecular clock mechanisms in the dental pulp might open doors for new therapeutic clinical strategies or improvement of already existing treatment possibilities. Echinomycin is a widely used inhibitor of HIF-1a signalling but has not yet been applied in analysis of the circadian clock [18, 19]
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