Deciphering the functions of Stromal Interaction Molecule-1 in amelogenesis using AmelX-iCre mice

Raed Said; Helyasadat Mortazavi; David Cooper; Katie Ovens; Ian McQuillan; Silvana Papagerakis; Petros Papagerakis

doi:10.3389/fphys.2023.1100714

Deciphering the functions of Stromal Interaction Molecule-1 in amelogenesis using AmelX-iCre mice

Front Physiol. 2023 Mar 1:14:1100714. doi: 10.3389/fphys.2023.1100714. eCollection 2023.

Authors

Raed Said^{1

2}, Helyasadat Mortazavi^{1

2}, David Cooper², Katie Ovens³, Ian McQuillan⁴, Silvana Papagerakis^{5

6}, Petros Papagerakis^{1

2}

Affiliations

¹ College of Dentistry, University of Saskatchewan, Saskatoon, SK, Canada.
² Department of Anatomy, Physiology and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
³ Department of Computer Science, University of Calgary, Calgary, AB, Canada.
⁴ Department of Computer Sciences, College of Arts and Sciences, University of Saskatchewan, Saskatoon, SK, Canada.
⁵ Department of Surgery, College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada.
⁶ Department of Otolaryngology-Head and Neck Surgery, School of Medicine, University of Michigan, Ann Arbor, MI, United States.

Abstract

Introduction: The intracellular Ca2+ sensor stromal interaction molecule 1 (STIM1) is thought to play a critical role in enamel development, as its mutations cause Amelogenesis Imperfecta (AI). We recently established an ameloblast-specific (AmelX-iCre) Stim1 conditional deletion mouse model to investigate the role of STIM1 in controlling ameloblast function and differentiation in vivo (Stim1 cKO). Our pilot data (Said et al., J. Dent. Res., 2019, 98, 1002-1010) support our hypothesis for a broad role of Stim1 in amelogenesis. This paper aims to provide an in-depth characterization of the enamel phenotype observed in our Stim1 cKO model. Methods: We crossed AmelX-iCre mice with Stim1-floxed animals to develop ameloblast-specific Stim1 cKO mice. Scanning electron microscopy, energy dispersive spectroscopy, and micro- CT were used to study the enamel phenotype. RNAseq and RT-qPCR were utilized to evaluate changes in the gene expression of several key ameloblast genes. Immunohistochemistry was used to detect the amelogenin, matrix metalloprotease 20 and kallikrein 4 proteins in ameloblasts. Results: Stim1 cKO animals exhibited a hypomineralized AI phenotype, with reduced enamel volume, diminished mineral density, and lower calcium content. The mutant enamel phenotype was more severe in older Stim1 cKO mice compared to younger ones and changes in enamel volume and mineral content were more pronounced in incisors compared to molars. Exploratory RNAseq analysis of incisors' ameloblasts suggested that ablation of Stim1 altered the expression levels of several genes encoding enamel matrix proteins which were confirmed by subsequent RT-qPCR. On the other hand, RT-qPCR analysis of molars' ameloblasts showed non-significant differences in the expression levels of enamel matrix genes between control and Stim1-deficient cells. Moreover, gene expression analysis of incisors' and molars' ameloblasts showed that Stim1 ablation caused changes in the expression levels of several genes associated with calcium transport and mitochondrial kinetics. Conclusions: Collectively, these findings suggest that the loss of Stim1 in ameloblasts may impact enamel mineralization and ameloblast gene expression.

Keywords: ameloblast; amelogenesis; calcium; enamel; mineralised tissue; store operated Ca 2+ entry; stromal interacting molecule 1 (STIM1).

Grants and funding

This work was partially funded by the University of Saskatchewan College of Dentistry start-up funds to PP and by the University of Saskatchewan College of Medicine and Vice Provost Office start-up funds to SP.