United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
Mol Cell Biochem. 2021 Oct;476(10):3869-3877. doi: 10.1007/s11010-021-04187-1. Epub 2021 Jun 15.
Brefeldin A (BFA) disrupts the structure of the Golgi apparatus to trigger ER stress signaling pathways. On the other hand, treatment with BFA induces the activation of CREB3, the protein structure of which is similar to that of ATF6. In this study, we established Neuro2a cells in which three different transcription factors, namely, ATF4, ATF3 and CREB3, were deficient using the CRISPR/Cas9 approach, and we investigated the BFA-induced ER and Golgi stress response in these cells. BFA treatment rapidly induced ATF4, ATF3, Herp and GADD153 protein expression in Neuro2a cells. ATF4-deficient Neuro2a cells exhibited significantly decreased mRNA and protein expression of ATF3 and Herp but not GADD153; however, cells deficient in ATF3 exhibited minimal effects on GADD34, GADD153 and Herp expression. The cleavage of CREB3 in Neuro2a cells was triggered by BFA; however, the expression of several ER and Golgi stress-related factors was hardly influenced by the CREB3 deficiency in these Neuro2a cells. This study shows that CREB3 minimally associates with typical ER stress-inducible responses in Neuro2a cells. Therefore, identification and characterization of the downstream transcriptional targets of CREB3 is required to clarify not only Golgi stress response but also its relationship with ER stress signaling pathways.
布雷菲德菌素 A（BFA）破坏高尔基体的结构，从而触发内质网应激信号通路。另一方面，BFA 的处理会诱导 CREB3 的激活，其蛋白结构与 ATF6 相似。在本研究中，我们使用 CRISPR/Cas9 方法建立了三种不同转录因子（即 ATF4、ATF3 和 CREB3）缺失的 Neuro2a 细胞，并研究了这些细胞中 BFA 诱导的内质网和高尔基体应激反应。BFA 处理可迅速诱导 Neuro2a 细胞中 ATF4、ATF3、Herp 和 GADD153 蛋白的表达。ATF4 缺失的 Neuro2a 细胞中 ATF3 和 Herp 的 mRNA 和蛋白表达显著降低，但 GADD153 表达不受影响；然而，ATF3 缺失的细胞对 GADD34、GADD153 和 Herp 的表达影响最小。BFA 可触发 Neuro2a 细胞中 CREB3 的切割；然而，在这些 Neuro2a 细胞中 CREB3 的缺失几乎不影响几种内质网和高尔基体应激相关因子的表达。本研究表明，CREB3 与 Neuro2a 细胞中典型的内质网应激诱导反应的关联最小。因此，需要鉴定和表征 CREB3 的下游转录靶标，以不仅阐明高尔基体应激反应，而且阐明其与内质网应激信号通路的关系。
