Elucidation of brefeldin A-induced ER and Golgi stress responses in Neuro2a cells.

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.

Regulation of the ER-bound transcription factor Luman/CREB3 in HEK293 cells.

CREB3 is a transcription factor localized to the ER. Here, we investigated endogenous CREB3 expression in HEK293 cells using pharmacological and genome editing approaches. Full-length CREB3 detected under resting conditions disappeared following treatment with tunicamycin, brefeldin A and nigericin.

Elucidating post-translational regulation of mouse CREB3 in Neuro2a cells.

CREB3 is an ER membrane-bound transcription factor; however, post-translational regulation of CREB3, including expression, processing, and activation, is not fully characterized. We therefore constructed several types of mouse CREB3 expression genes and elucidated their expression in Neuro2a cells by treatment with stimuli and co-transfection with genes associated with ER-Golgi homeostasis, such as mutant Sar1 [H79G], GRP78, and KDEL receptor 1 (KDELR1). Interestingly, treatment of Neuro2a cells expressing Flag-tagged full-length CREB3 with monensin and nigericin induced the expression of the approximately 50 kDa N-terminal fragment; however, its cleavage was not parallel to the levels of GADD153 and LC3-II.