RESUMO
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.
Assuntos
Brefeldina A/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Complexo de Golgi/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/genética , Complexo de Golgi/genética , Camundongos , Transdução de Sinais/genéticaRESUMO
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. Co-transfection of full-length CREB3 together with Sar1 [H79G], GRP78, or KDELR1 showed that only Sar1 [H79G] induced expression of the cleaved form, and KDELR1 dramatically decreased the expression of the full-length form. Accordingly, Sar1 [H79G]- and KDELR1-overexpression influenced GAL4-CREB3-dependent luciferase activities. To understand the activation of CREB3 under more pathophysiological conditions, we focused on the effect of metal ions on CREB3 cleavage in Neuro2a cells. Among the six metal ions we tested, only copper ion stabilized full-length CREB3 expression. Copper ion also increased its N-terminal form and GAL4-CREB3-dependent luciferase activity, which was accompanied by the increase in the ubiquitinated proteins in Neuro2a cells. Taken together, CREB3 expression is regulated by multiple ER-Golgi resident factors in a post-translational manner, but its processing is not directly associated with ER stress and autophagic dysfunction. This finding is especially true for the unique action of the copper ion on CREB3 stabilization and processing in parallel to aberration of ubiquitin-proteasome system, which might provide new insights into understanding the mechanisms of intractable disorders.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Animais , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Chaperona BiP do Retículo Endoplasmático , Complexo de Golgi/genética , Complexo de Golgi/patologia , CamundongosRESUMO
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. Treatment with cycloheximide and MG132 indicated that endogenous CREB3 is a proteasome substrate. Using cells deficient for the ER-associated protein degradation (ERAD) factors SEL1L and Herp, we demonstrate that SEL1L, but not Herp, plays a crucial role in the posttranslational regulation of full-length CREB3 expression. In addition, kifunensine, an α-mannosidase inhibitor, remarkably increased full-length CREB3 expression. Our study suggests that endogenous full-length CREB3 is a novel substrate for ERAD and identifies unique cellular signals distinct from those in canonical ER stress.