STIM1 a calcium sensor promotes the assembly of an ECM that contains Extracellular vesicles and factors that modulate mineralization

Abstract

Osteoblasts and odontoblasts, are non-excitable cells and facilitate mass calcium transport during matrix mineralization. A sophisticated Ca2+ sensing mechanism is used to maintain Ca2+ homeostasis. STIM1 (Stromal interaction molecule 1) is a calcium sensor protein localized in the ER membrane and maintains calcium homeostasis by initiating the store-operated Ca2+ entry (SOCE) process, following store depletion. The role of STIM1 in dentin mineralization is yet to be elucidated. Therefore, transgenic DPSCs were generated in which overexpression or knockdown of STIM1 was achieved to study its function in matrix mineralization. Gene expression analysis and Alizarin Red staining assay demonstrated upregulation of genes involved in odontogenic differentiation and matrix mineralization with increased calcium deposition with STIM1 overexpression. Topology of the ECM examined by Field Emission Scanning Electron Microscopy (FESEM) showed the presence of large amounts of extracellular microvesicles with mineral deposits. Interestingly, silencing STIM1 resulted in fewer vesicles and less mineral deposits in the ECM. Analysis of the dentin-pulp complex of STIM1- deficient mice by micro-CT show reduced dentin thickness, malformed and highly porous alveolar bone, suggesting a cell intrinsic role for STIM1 in dentin mineralization. Confocal microscopy showed that DMP1-mediated depletion of store Ca2+ resulted in aggregation or “puncta-formation” of STIM1 at the plasma membrane indicative of a gating arrangement with Orai1 for Ca2+ influx. Together, our data provide evidence for an important role for STIM1 in dentin and alveolar bone mineralization by influencing intracellular Ca2+ oscillations that could provide signals for a wide array of cellular functions. Statement of SignificanceCalcium signaling and transport are fundamental to bone and dentin mineralization. Osteoblasts and odontoblasts transport large amounts of Ca2+ to the extracellular matrix. These cells maintain calcium homeostasis by spatially distributed calcium pumps and channels at the plasma membrane. STIM1 an ER Ca2+ sensor protein is an important component of the store-operated calcium entry (SOCE) process. In this study, we examined the role of STIM1 during the differentiation of dental pulp stem cells into functional odontoblasts and formation of mineralized dentin matrix. Stimulation of these cells with DMP1, a key regulatory protein in matrix mineralization, stimulates STIM1-mediated release of ER Ca2+ and SOCE activation. Silencing of STIM1 impairs signaling events, release of exosomes containing matrix proteins and matrix mineralization.