<b>Effect of 1,25-dihydroxyvitamin D</b><b>₃</b><b> on spontaneous calcium responses in rat dental epithelial SF2 cells revealed by long-term </b><b>imaging </b>

Abstract

Genetically encoded calcium indicators (GECIs) are suitable for long-term imaging studies. In this study, we employed a highly sensitive GECI, G-GECO, and achieved efficient gene delivery with an adenoviral vector. The adenoviral vector allowed us to express G-GECO in more than 80% of cells. More than 80% of G-GECO-expressing cells showed an ATP-induced increase in fluorescence intensity due to Ca 2+ release from intracellular stores and subsequent Ca 2+ entry. The fluorescence intensity of these cells was increased more than 2-fold by stimulation with 10 μM ATP. We applied long-term imaging (for ~10 h) to monitor Ca 2+ responses in SF2, a rat dental epithelial cell line, in culture conditions. SF2 cells showed intermittent rises in the intracellular Ca 2+ concentration in the presence of 100 nM 1,25-dihydroxyvitamin D 3 . Many of these Ca 2+ responses began at a specific location in the cytoplasm and spread throughout the entire cytoplasm. The combination of efficient gene delivery with an adenoviral vector and long-term imaging with a highly sensitive GECI enabled detection of intermittent Ca 2+ responses that occur only 3-10 times/h/ 100 cells. This method could be useful to study the effects of Ca 2+ responses for regulating longterm processes, such as gene expression, cell migration, and cell division, in many cell types.The hormonally active form of vitamin D 3 , 1,25-dihydroxyvitamin D 3 (VD3), is associated with calcium and phosphorus homeostasis, and maintains bone content through effects on the intestine, bone, kidney, and parathyroid gland (13). In addition to these traditional calcemic activities, VD3 is involved in cell proliferation, differentiation, and immunomodulation (11). Many of the biological actions of VD3 are mediated by the nuclear vitamin D receptor (VDR), a ligand-dependent transcription factor belonging to the superfamily of steroid/thyroid hormone receptors (13). Moreover, VD3 modulates the intracellular Ca 2+ concentration ([Ca 2+

Spontaneous calcium responses of SF2 rat dental epithelial cells stably expressing the calcium sensor G-GECO

“…Unlike chemically synthesized fluorescent Ca 2+ indicators, GECIs do not leak out of cells or accumulate in organelles, meaning they are suitable for long-term imaging studies under cell culture conditions (Tanimura 2015). We previously reported vitamin D3-induced spontaneous Ca 2+ responses of SF2 cells by performing long-term Ca 2+ imaging of SF2 cells that expressed G-GECO using adenovirus vectors (Murata et al 2016). Despite the high efficiency of gene transfer, adenovirus vectors are relatively cytotoxic and the delivered genes are transiently expressed (Russell 2000).…”