Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China; Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China; Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China.
Cell Signal. 2019 May;57:89-101. doi: 10.1016/j.cellsig.2019.01.008. Epub 2019 Jan 28.
Endoplasmic reticulum (ER) stress and circadian clockwork signaling pathways mutually regulate various cellular functions, but the details regarding the cross-talk between these pathways in mammalian cells are unclear. In this study, whether perturbation of ER stress signaling affects the cellular circadian clockwork and transcription of clock-controlled genes was investigated in NIH3T3 mouse fibroblasts. An NIH3T3 cell model stably expressing luciferase (Luc) under the control of the Bmal1 clock gene promoter was established using a lentiviral system. Then, Luc activity was monitored in real-time to detect Bmal1-Luc oscillations. The ER stress activators thapsigargin (Tg) and tunicamycin (Tm) markedly reduced Bmal1-Luc oscillation amplitudes and induced phase delay shifts in NIH3T3 cells. Treatment with Tg/Tm activated ER stress signaling by upregulating GRP78, CHOP, ATF6, and ATF4 and simultaneously significantly decreased BMAL1 protein levels and inhibited the transcription of circadian clock (Bmal1, Per2, Nr1d1, and Dbp) and clock-controlled (Scad1, Fgf7, and Arnt) genes. 4-Phenylbutyric acid, an ER stress inhibitor, alleviated the transcriptional repression of the circadian clock genes and partially restored Bmal1-Luc oscillation amplitudes in Tg- or Tm-treated NIH3T3 cells. More importantly, knock-down of ATF4, but not ATF6, in Tg-treated NIH3T3 cells partially rescued Bmal1-Luc oscillation amplitudes and mRNA expression of the four circadian clock genes. Taken together, our study demonstrates that ER stress activation inhibits the transcription of circadian clock and clock-controlled genes via an ATF4-dependent mechanism.
内质网（ER）应激和生物钟信号通路相互调节各种细胞功能，但哺乳动物细胞中这些通路之间的串扰细节尚不清楚。在这项研究中，研究了 ER 应激信号转导的扰动是否会影响细胞生物钟和时钟控制基因的转录。使用慢病毒系统建立了稳定表达受 Bmal1 时钟基因启动子控制的荧光素酶（Luc）的 NIH3T3 小鼠成纤维细胞模型。然后，实时监测 Luc 活性以检测 Bmal1-Luc 振荡。ER 应激激活剂他普西龙（Tg）和衣霉素（Tm）显著降低了 Bmal1-Luc 振荡幅度，并诱导 NIH3T3 细胞发生相位延迟。用 Tg/Tm 处理通过上调 GRP78、CHOP、ATF6 和 ATF4 激活 ER 应激信号，同时显著降低 BMAL1 蛋白水平并抑制生物钟（Bmal1、Per2、Nr1d1 和 Dbp）和时钟控制（Scad1、Fgf7 和 Arnt）基因的转录。ER 应激抑制剂 4-苯基丁酸减轻了 Tg 或 Tm 处理的 NIH3T3 细胞中生物钟基因的转录抑制作用，并部分恢复了 Bmal1-Luc 振荡幅度。更重要的是，在 Tg 处理的 NIH3T3 细胞中敲低 ATF4，但不是 ATF6，部分挽救了 Bmal1-Luc 振荡幅度和四个生物钟基因的 mRNA 表达。总之，我们的研究表明，ER 应激激活通过依赖 ATF4 的机制抑制生物钟和时钟控制基因的转录。
