Role of the store-operated calcium entry on differentiation of dental epithelial cells

抄録

[Background]

Change in intracellular calcium concentration ([Ca²⁺]_i) is a key signal for regulation of a wide range of biological processes, including cell differentiation and migration. Recent studies have shown that amelogenesis imperfecta is caused by the deficiency of store-operated calcium entry (SOCE), the main calcium influx mechanisms in non-excitable cells. These findings suggest important roles of SOCE on amelogenesis. We visualized calcium responses in rat dental epithelial cells (SF2) and human dental pulp stem cell (DPSC) using genetically encoded calcium indicators, and examined the role of SOCE on the cell migration and gene expression.

[Methods]

Ca²⁺ responses and cell migrations were visualized simultaneously with epifluorescent microscopy and TIRF microscopy using the fluorescent imaging system consist of Nikon TE2000 and EM-CCD camera. The Ca²⁺ sensor protein, YC-Nano50, G-GECO or R-GECO, were expressed using adenoviral vector or lentiviral vector. Quantitative analysis of the cell migration was performed using Oris^TM cell migration assay. Change of gene expression was analyzed using comprehensive analysis using next generation sequencing, RT-PCR and qPCR.

　[Results]

SF2 showed intermittent rises in [Ca²⁺]_i during the long time live-cell-imaging in a cell culture condition. These rises in [Ca²⁺]_i were reduced by SOCE inhibitor, LaCl₃. TIRF microscopy showed a formation and active movement of cell protrusions in the presence of serum or EGF plus CXCL12. The addition of LaCl₃ decreased basal [Ca²⁺]_i, and subsequently reduced serum-dependent cell protrusions. Quantitative analyses indicated that LaCl₃ inhibited serum-dependent cell migration almost completely. These results suggest SOCE regulates cell migration for morphogenesis of dental epithelial cells.

We also found that the frequency of the calcium responses and motility of SF2 cells was drastically enhanced by the co-culture with DPSC. LaCl₃ also reduced the rises in [Ca²⁺]_i in the cell co-culture condition of SF2 and DPSC. Comprehensive gene expression analyses suggested the enhanced expression of TGF-β related genes with this co-culture. The inhibition of SOCE also changed expressions of some genes. We are trying to clarify mechanisms for the co-culture-mediated Ca²⁺ responses and SOCE-mediated gene expression.

[Conclusions]

Our results suggest essential roles of SOCE-mediated Ca²⁺ responses in the tooth development, through controlling cell migrations and gene expression.