Institute of Transformative Bio-Molecules (ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
Laboratory of Animal Integrative Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
Sci Rep. 2022 Oct 15;12(1):17325. doi: 10.1038/s41598-022-22166-4.
Real-time monitoring of cellular temperature responses is an important technique in thermal biology and drug development. Recent study identified that Na/Ca exchanger (NCX)-dependent Ca 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-dependent transcription in living cells, we developed a luciferase-based real-time reporting system by using Per1 promoter, Per2 promoter, Ca/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 signaling which can be applied to evaluation of drugs.
实时监测细胞温度反应是热生物学和药物开发中的一项重要技术。最近的研究表明，钠/钙交换器（NCX）依赖性钙内流将冷信号转导到哺乳动物培养细胞的生物钟。这一发现提出了一个观点，即可以通过监测时钟基因表达来分析细胞对冷信号的反应。我们发现，与在 37°C 培养相比，在 Rat-1 成纤维细胞中在 27°C 下培养时 Per1 和 Per2 上调。为了在活细胞中监测冷-Ca 依赖性转录，我们使用 Per1 启动子、Per2 启动子、Ca/cAMP 反应元件 (CRE) 或 NFAT 结合元件开发了基于荧光素酶的实时报告系统。我们发现苯并氧苯基 NCX 抑制剂 KB-R7943 和 SN-6 但不是 SEA-0400 或 YM-244769 抑制了 Per2 的冷诱导。我们的研究建立了一种实时监测冷 Ca 信号的系统，可用于评估药物。
