Table_2_Calcium Sets the Clock in Ameloblasts.DOCX

BackgroundStromal interaction molecule 1 (STIM1) is one of the main components of the store operated Ca2+ entry (SOCE) signaling pathway. Individuals with mutated STIM1 present severely hypomineralized enamel characterized as amelogenesis imperfecta (AI) but the downstream molecular mechanisms involved remain unclear. Circadian clock signaling plays a key role in regulating the enamel thickness and mineralization, but the effects of STIM1-mediated AI on circadian clock are unknown.ObjectivesThe aim of this study is to examine the potential links between SOCE and the circadian clock during amelogenesis.MethodsWe have generated mice with ameloblast-specific deletion of Stim1 (Stim1fl/fl/Amelx-iCre+/+, Stim1 cKO) and analyzed circadian gene expression profile in Stim1 cKO compared to control (Stim1fl/fl/Amelx-iCre–/–) using ameloblast micro-dissection and RNA micro-array of 84 circadian genes. Expression level changes were validated by qRT-PCR and immunohistochemistry.ResultsStim1 deletion has resulted in significant upregulation of the core circadian activator gene Brain and Muscle Aryl Hydrocarbon Receptor Nuclear Translocation 1 (Bmal1) and downregulation of the circadian inhibitor Period 2 (Per2). Our analyses also revealed that SOCE disruption results in dysregulation of two additional circadian regulators; p38α mitogen-activated protein kinase (MAPK14) and transforming growth factor-beta1 (TGF-β1). Both MAPK14 and TGF-β1 pathways are known to play major roles in enamel secretion and their dysregulation has been previously implicated in the development of AI phenotype.ConclusionThese data indicate that disruption of SOCE significantly affects the ameloblasts molecular circadian clock, suggesting that alteration of the circadian clock may be partly involved in the development of STIM1-mediated AI.

背景：背景基质相互作用分子1（STIM1）是储存操作钙离子进入（SOCE）信号通路的主要成分之一。STIM1基因突变个体表现出严重低矿化牙釉质，其特征为牙本质发育不全（AI），然而涉及其中的下游分子机制尚不明确。昼夜节律时钟信号在调节牙釉质厚度和矿化过程中发挥着关键作用，但STIM1介导的AI对昼夜节律时钟的影响尚不清楚。目标：本研究旨在探讨SOCE与牙本质发育过程中昼夜节律之间的潜在联系。方法：我们通过Amelx-iCre+/+小鼠特异性删除Stim1基因（Stim1fl/fl/Amelx-iCre+/+，Stim1 cKO）并分析Stim1 cKO与对照（Stim1fl/fl/Amelx-iCre–/–）在牙本质细胞中84个昼夜节律基因的表达谱，使用牙本质细胞显微切割和RNA微阵列。通过定量逆转录聚合酶链反应（qRT-PCR）和免疫组织化学验证了表达水平的变化。结果：Stim1基因删除导致核心昼夜节律激活基因脑和肌肉芳香烃受体核转位1（Bmal1）的上调以及昼夜节律抑制因子Period 2（Per2）的下调。我们的分析还揭示了SOCE破坏导致两个额外的昼夜节律调节因子失调；p38α丝裂原活化蛋白激酶（MAPK14）和转化生长因子-β1（TGF-β1）。已知MAPK14和TGF-β1途径在牙釉质分泌中发挥着主要作用，其失调已被先前暗示与AI表型的发生有关。结论：这些数据表明，SOCE的破坏显著影响了成牙本质细胞的分子昼夜节律时钟，表明昼夜节律时钟的改变可能部分参与了STIM1介导的AI的发生。