Abstract
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.
Highlights
Calcium plays a pivotal role in enamel mineralization, yet the exact mechanisms involved in Ca2+ transport and the roles of Ca2+ signaling in regulating ameloblast functions during amelogenesis remain unclear (Berdal et al, 1995; BailleulForestier et al, 1996; Nurbaeva et al, 2017)
We found that fourteen circadian genes were significantly differentially expressed (P < 0.05) in Stim1 cKO ameloblasts with at least a twofold differential expression compared to control (Figures 1B,C, Supplementary Table 2, and Supplementary Datasheet 1)
We found that Stim1 disruption had led to significant changes in the expression levels of several key clock genes that form the circadian loops, including Bmal1, Period 2 (Per2), Fbxl3, Ror-a, and Ror-c, which strongly indicates that Store-operated Ca2+ entry (SOCE) may influence the ameloblast circadian clock system
Summary
Calcium plays a pivotal role in enamel mineralization, yet the exact mechanisms involved in Ca2+ transport and the roles of Ca2+ signaling in regulating ameloblast functions during amelogenesis remain unclear (Berdal et al, 1995; BailleulForestier et al, 1996; Nurbaeva et al, 2017). Several subsequent reports have shown that Ca2+ transport occurs principally transcellularly and mainly through high capacity intracellular stores in endoplasmic reticulum (ER) (Hubbard, 2000; Lacruz et al, 2012b; Nurbaeva et al, 2015a,b, 2017). This mode of transport is termed Store-operated Ca2+ entry (SOCE) and is mainly mediated by the ER transmembrane proteins Stromal interaction molecule 1 and 2 (STIM1 and STIM2) and the highly selective plasma membrane (PM) calcium release activated channels (CRAC) (Prakriya and Lewis, 2015; Lacruz, 2017). 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
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