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

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Abstract

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.

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Data availability

The data generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ATF3:

Activating transcription factor 3

ATF4:

Activating transcription factor 4

ATF6:

Activating transcription factor 6

CREB3:

cAMP response element binding protein 3

CRISPR/Cas9:

Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 9

ER:

Endoplasmic reticulum

ERAD:

ER stress-associated degradation

ERSE:

ER stress response element

GADD153:

Growth arrest and DNA damage inducible gene 153

GRP78:

78 KDa glucose-regulated protein

G3PDH:

Glyceraldehyde 3-phosphate dehydrogenase

Herp:

Homocysteine-induced ER protein

IRE1:

Inositol-requiring enzyme-1

PERK:

PKR-like endoplasmic reticulum kinase

TFE3:

Transcription factor E3

RT-PCR:

Reverse transcription polymerase chain reaction

XBP1:

X-box binding protein 1

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Acknowledgements

This work is, in part, supported by Grant-in-aid from the Japan Society for the Promotion of Science (JSPS, Japan, KAKENHI, No. 19H04030 to K.O.).

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Authors and Affiliations

  1. United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi & Yoko Hirata

  2. Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi, Tomoyuki Hasegawa, Kanto Takahashi & Yoko Hirata

  3. Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi, Yuri Mizutani & Yoko Hirata

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  1. Kentaro Oh-hashi
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  2. Tomoyuki Hasegawa
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  4. Kanto Takahashi
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Contributions

KO, TH and KT discussed and designed the research; KO, TH, YM and KT performed experiments; KO and YH confirmed the results; KO and TH prepared the manuscript.

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Correspondence to Kentaro Oh-hashi.

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Oh-hashi, K., Hasegawa, T., Mizutani, Y. et al. Elucidation of brefeldin A-induced ER and Golgi stress responses in Neuro2a cells. Mol Cell Biochem 476, 3869–3877 (2021). https://doi.org/10.1007/s11010-021-04187-1

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