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Genome-wide mRNA profiling identifies RCAN1 and GADD45A as regulators of the transitional switch from survival to apoptosis during ER stress.
The FEBS Journal ( IF 5.5 ) Pub Date : 2019-12-27 , DOI: 10.1111/febs.15195 Rafal Bartoszewski 1 , Magdalena Gebert 1 , Anna Janaszak-Jasiecka 1 , Aleksandra Cabaj 2 , Jarosław Króliczewski 1 , Sylwia Bartoszewska 3 , Aleksandra Sobolewska 1 , David K Crossman 4 , Renata Ochocka 1 , Wojciech Kamysz 3 , Leszek Kalinowski 5 , Michał Dąbrowski 2 , James F Collawn 6
The FEBS Journal ( IF 5.5 ) Pub Date : 2019-12-27 , DOI: 10.1111/febs.15195 Rafal Bartoszewski 1 , Magdalena Gebert 1 , Anna Janaszak-Jasiecka 1 , Aleksandra Cabaj 2 , Jarosław Króliczewski 1 , Sylwia Bartoszewska 3 , Aleksandra Sobolewska 1 , David K Crossman 4 , Renata Ochocka 1 , Wojciech Kamysz 3 , Leszek Kalinowski 5 , Michał Dąbrowski 2 , James F Collawn 6
Affiliation
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Endoplasmic reticulum (ER) stress conditions promote a cellular adaptive mechanism called the unfolded protein response (UPR) that utilizes three stress sensors, inositol‐requiring protein 1, protein kinase RNA‐like ER kinase, and activating transcription factor 6. These sensors activate a number of pathways to reduce the stress and facilitate cell survival. While much is known about the mechanisms involved that modulate apoptosis during chronic stress, less is known about the transition between the prosurvival and proapoptotic factors that determine cell fate. Here, we employed a genetic screen that utilized three different pharmacological stressors to induce ER stress in a human‐immortalized airway epithelial cell line, immortalized human bronchial epithelial cells. We followed the stress responses over an 18‐h time course and utilized real‐time monitoring of cell survival, next‐generation sequencing, and quantitative real‐time PCR to identify and validate genes that were upregulated with all three commonly employed ER stressors, inhibitor of calpain 1, tunicamycin, and thapsigargin. growth arrest and DNA damage‐inducible alpha (GADD45A ), a proapoptotic factor, and regulator of calcineurin 1 (RCAN1 ) mRNAs were identified and verified by showing that small interfering RNA (siRNA) knockdown of GADD45A decreased CCAAT‐enhancer‐binding protein homologous protein (a.k.a DDIT3 ), BCL2‐binding component 3 (a.k.a. BBC3), and phorbol‐12‐myristate‐13‐acetate‐induced protein 1 expression, 3 proapoptotic factors, and increased cell viability during ER stress conditions, whereas siRNA knockdown of RCAN 1 dramatically decreased cell viability. These results suggest that the relative levels of these two genes regulate cell fate decisions during ER stress independent of the type of ER stressor.
中文翻译：
全基因组mRNA谱鉴定RCAN1和GADD45A为内质网应激期间从存活到凋亡的过渡性转换的调节因子。
内质网（ER）胁迫条件促进了一种称为未折叠蛋白应答（UPR）的细胞适应性机制，该机制利用三个应激传感器，即需要肌醇的蛋白质1，蛋白激酶RNA样的ER激酶和激活转录因子6。减少压力并促进细胞存活的多种途径。尽管对慢性应激过程中调节细胞凋亡的机制了解很多，但对决定细胞命运的生存因子和促凋亡因子之间的转换了解的很少。在这里，我们采用了一种遗传筛选方法，该方法利用三种不同的药理学应激源在人类永生化气道上皮细胞系，永生化人支气管上皮细胞系中诱导内质网应激。我们在18小时的时间过程中跟踪了应激反应，并利用细胞存活的实时监控，下一代测序和定量实时PCR来鉴定和验证被三种常用的ER应激因子，抑制剂上调的基因钙蛋白酶1，衣霉素和毒胡萝卜素。生长停滞和DNA损伤诱导α（通过显示GADD45A的小干扰RNA（siRNA）敲低可降低CCAAT-增强子结合蛋白同源蛋白（aka DDIT3），BCL2结合成分，从而鉴定并验证了GADD45A），促凋亡因子和钙调神经磷酸酶1（RCAN1）mRNA的调节剂。3（aka BBC3），以及佛波醇12-肉豆蔻酸酯-13-乙酸酯诱导的蛋白1表达，3个促凋亡因子和内质网应激条件下细胞活力的提高，而RCAN 1的siRNA敲低显着降低了细胞活力。这些结果表明，这两个基因的相对水平在ER应激期间调节细胞命运的决定，而与ER应激源的类型无关。
更新日期：2019-12-27
中文翻译：
全基因组mRNA谱鉴定RCAN1和GADD45A为内质网应激期间从存活到凋亡的过渡性转换的调节因子。
内质网（ER）胁迫条件促进了一种称为未折叠蛋白应答（UPR）的细胞适应性机制，该机制利用三个应激传感器，即需要肌醇的蛋白质1，蛋白激酶RNA样的ER激酶和激活转录因子6。减少压力并促进细胞存活的多种途径。尽管对慢性应激过程中调节细胞凋亡的机制了解很多，但对决定细胞命运的生存因子和促凋亡因子之间的转换了解的很少。在这里，我们采用了一种遗传筛选方法，该方法利用三种不同的药理学应激源在人类永生化气道上皮细胞系，永生化人支气管上皮细胞系中诱导内质网应激。我们在18小时的时间过程中跟踪了应激反应，并利用细胞存活的实时监控，下一代测序和定量实时PCR来鉴定和验证被三种常用的ER应激因子，抑制剂上调的基因钙蛋白酶1，衣霉素和毒胡萝卜素。生长停滞和DNA损伤诱导α（通过显示GADD45A的小干扰RNA（siRNA）敲低可降低CCAAT-增强子结合蛋白同源蛋白（aka DDIT3），BCL2结合成分，从而鉴定并验证了GADD45A），促凋亡因子和钙调神经磷酸酶1（RCAN1）mRNA的调节剂。3（aka BBC3），以及佛波醇12-肉豆蔻酸酯-13-乙酸酯诱导的蛋白1表达，3个促凋亡因子和内质网应激条件下细胞活力的提高，而RCAN 1的siRNA敲低显着降低了细胞活力。这些结果表明，这两个基因的相对水平在ER应激期间调节细胞命运的决定，而与ER应激源的类型无关。
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