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

生物钟是一个高度保守的系统，存在于大脑和许多外周组织中，负责在基因和蛋白质表达中产生24小时节律，并协调分子途径、细胞和组织的时间组织。生物钟基因在形成牙釉质的成釉细胞中高度表达，但生物钟基因在成釉细胞分化中的确切作用尚不清楚。成釉细胞分化在很大程度上也取决于细胞内钙（iCa2+）信号。基质相互作用分子1 （STIM1）是内质网钙离子浓度的主要传感器。内质网钙储存的消耗激活STIM1蛋白，进而结合并打开主要由ORAI钙释放激活钙调节剂1 （ORAI1）蛋白形成的质膜中的钙通道。iCa2+如何调节成釉细胞特异性基因尚不清楚。我们最近的研究表明，在成釉细胞中，生物钟和iCa2+信号之间存在直接的相互作用。该项目将验证时钟基因通过在24小时周期内调节基因表达来控制成釉细胞分化和功能的假设。我们还将测试生物钟在内部控制调节iCa2+中起关键作用的假设。为了验证我们的假设，我们将在体外和体内结合使用昼夜节律生物学方法。我们的目标是：1；利用时钟基因报告小鼠、时钟基因敲除（KO）小鼠和细胞培养实验等遗传工具，表征昼夜节律振荡器在调节成釉细胞分化、基因表达和细胞内钙（iCa2+）信号传导中的昼夜节律特征和作用。2. 评估环境因素（光照和黑暗周期）在成釉细胞分化过程中设置和维持昼夜节律基因和iCa2+振荡的作用。我们的项目意义重大，因为它专注于两个完全未被研究但至关重要的方面，即昼夜节律时钟效应和iCa2+的作用。这项研究是了解昼夜节律时钟如何在成釉细胞分化中发挥关键作用以及昼夜节律振荡如何控制细胞功能的第一步。这项研究将在昼夜生物学、细胞分化、矿化和器官生理学领域开辟一个新的领域。

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.