Real time monitoring of cold Ca 2+ dependent transcription and its modulation by NCX inhibitors

Real-time monitoring of cellular temperature responses is an important technique in thermal biology and drug development. Recent study identified that Na + /Ca 2+ exchanger (NCX)-dependent Ca 2+ influx transduces cold signals to circadian clock in mammalian cultured cells. The finding raised an idea that cellular responses to the cold signals can be analyzed by monitoring of clock gene expression. We found that Per1 and Per2 were up-regulated after culture at 27 °C compared to 37 °C in Rat-1 fibroblasts. In order to monitor cold-Ca 2+ -dependent transcription in living cells, we developed a luciferase-based real-time reporting system by using Per1 promoter , Per2 promoter, Ca 2+ /cAMP-response elements (CRE) or NFAT-binding elements. We found that benzyloxyphenyl NCX inhibitor KB-R7943 and SN-6, but not SEA-0400 or YM-244769 inhibited the cold induction of Per2 . Our study established a real-time monitoring system for cold Ca 2+ signaling which can be applied to evaluation of drugs.