Magnesium-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells by activating ERK/BMP2/Smads signaling

BACKGROUND: Magnesium (Mg(2+))-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells (DPSCs), but the regulatory mechanisms remain undefined. The aim of this work was to assess magnesium’s function in the above process and to explore the associated signaling pathway. METHODS: DPSCs underwent culture in odontogenic medium with the addition of 0, 1, 5, or 10 mM MgCl(2). Intracellular Mg(2+) levels in DPSCs were evaluated flow cytometrically using Mag-Fluo-4-AM. Mg(2+)-entry was inhibited by TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB). RNA-Sequencing was carried out for assessing transcriptome alterations in DPSCs during odontogenic differentiation associated with high extracellular Mg(2+). KEGG pathway analysis was performed to determine pathways related to the retrieved differentially expressed genes (DEGs). Immunoblot was performed for assessing magnesium’s role and exploring ERK/BMP2/Smads signaling. RESULTS: Mg(2+)-enriched microenvironment promoted odontogenic differentiation in DPSCs via intracellular Mg(2+) increase. Consistently, the positive effect of high extracellular Mg(2+) on odontogenic differentiation in DPSCs was blocked by 2-APB, which reduced Mg(2+) entry. RNA-sequencing identified 734 DEGs related to odontogenic differentiation in DPSCs in the presence of high extracellular Mg(2+). These DEGs participated in many cascades such as MAPK and TGF-β pathways. Consistently, ERK and BMP2/Smads pathways were activated in DPSCs treated with high extracellular Mg(2+). In agreement, ERK signaling inhibition by U0126 blunted the effect of high extracellular Mg(2+) on mineralization and odontogenic differentiation in DPSCs. Interestingly, BMP2, BMPR1, and phosphorylated Smad1/5/9 were significantly decreased by U0126, indicating that BMP2/Smads acted as downstream of ERK. CONCLUSIONS: Mg(2+)-enriched microenvironment promotes odontogenic differentiation in DPSCs by activating ERK/BMP2/Smads signaling via intracellular Mg(2+) increase. This study revealed that Mg(2+)-enriched microenvironment could be used as a new strategy for dental pulp regeneration.