ER-Ca²⁺ Sensors STIM1/2 are Important in Enamel Gene Expression

Objectives: Although the role of Ca²⁺ is well-established as a mineralizing agent in enamel, much less is known about the impact of changes in intracellular Ca²⁺ concentration in enamel mineralization. Mutations to the endoplasmic reticulum Ca²⁺ sensors STIM1 and STIM2 result in Amelogenesis Imperfecta. To determine the global impact of deleting STIM1 and STIM2 we generated a conditional KO mouse that does not express these proteins to perform RNA sequencing of enamel organ (EO) cells.
Methods: Three WT and Stim1/2-deficient mice each were analyzed for RNAseq. Total RNA was isolated from EO cells and quality was assessed on a Nanodrop 2000. RNAseq libraries were prepared using the TruSeq RNA sample prep v2 kit (Illumina). Amplified libraries were purified, quantified and visualized in an Agilent Tapestation 2200. Libraries were pooled equimolarly and loaded on the HiSeq 4000 Sequencing System (Illumina) and run as paired-end 150 nucleotide reads. Quality control of raw sequencing reads was performed using FastQC. Only paired-end (PE) reads were used. Trimmed PE reads were aligned to the mouse genome using TopHat v2.1. To calculate transcript abundances, we used Cufflinks v2.2.1 to convert raw reads to FPKM values. Differential expression analysis was performed using Cuffdiff. Differences in gene expression were considered statistically significant if p-value FDR < 0.01. Pathway enrichment analysis was performed using DAVID (NIH) and IPA (Qiagen) software considering DEG between WT and Stim1/2-deficient cells with adjusted p-value < 0.01 and absolute fold change > 2.
Results: A total of 370 genes were differentially expressed in Stim1/2-deficient mice EO cells with 114 significantly up-regulated and 256 genes significantly down-regulated (p < 0.01, absolute fold change ≥ 2). Among them we identified a number of enamel genes.
Conclusions: Stim1/2 are important regulators of enamel gene expression.