The circadian clock control of enamel formation: its role and regulatory links to the store-operated Ca2+ entry.

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The circadian clock is a highly-conserved system present in the brain and many peripheral tissues, responsible for producing 24-hour rhythms in gene and protein expression, and coordinating the temporal organization of molecular pathways, cells and tissues. Circadian clock genes are highly expressed in ameloblasts that form dental enamel, but the exact roles of the circadian clock genes in ameloblast differentiation are understudied. Ameloblast differentiation largely also depends upon the intracellular calcium (iCa2+) signaling. Stromal interaction molecule 1 (STIM1) is the main sensor of the calcium concentration in the endoplasmic reticulum. Depletion of endoplasmic reticulum calcium stores activates STIM1 protein which, in turn, binds and opens calcium channels in the plasma membrane formed mainly by ORAI Calcium Release-Activated Calcium Modulator 1 (ORAI1) protein. How iCa2+ regulate ameloblast specific genes remains unclear. Our most recent studies show evidence of a direct interplay between circadian clock and iCa2+ signaling in ameloblasts. This project will test the hypothesis that clock genes control ameloblast differentiation and function by modulating genes expression in 24hrs cycles. We will also test the hypothesis that the circadian clock plays a key role of internal control modulating iCa2+. To test our hypotheses, we will use a combination of circadian biology methods in vitro and in vivo. Our objectives are: 1. To characterize the circadian profile and roles of circadian oscillators in regulating ameloblast differentiation, gene expression and intracellular calcium (iCa2+) signaling using genetic tools i.e. clock genes reporter mice, clock genes knock out (KO) mice and cell culture assays. 2. To evaluate the role of environmental cues (light and dark cycles) in setting and keeping up circadian gene and iCa2+ oscillations during ameloblast differentiation. Our project is significant because it focuses on two completely understudied yet crucial aspects of amelogenesis, i.e. the circadian clock effects and the role of iCa2+. This study is the first step in understanding how the circadian clock plays a key role in ameloblast differentiation and how circadian oscillations control cell functions. This research will pioneer a new area in the field of circadian biology, cell differentiation, mineralization and organ physiology.