Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway.
Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway.
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DOI：
10.1042/bj20081706
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发表时间：
2009-02-01
期刊：
影响因子：
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通讯作者：
Kilberg MS
中科院分区：
文献类型：
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作者：
Gjymishka A;Su N;Kilberg MS
UPR（未折叠蛋白反应）途径由内质网应激传感器蛋白PERK [PKR（双链rna激活蛋白激酶）样ER激酶]、IRE1（肌醇要求激酶1）和ATF6（激活转录因子6）介导的三个信号级联组成。本研究表明，在UPR激活剂thapsigarin和tunicamycin处理的HepG2细胞中，ASNS（天冬酰胺合成酶）转录活性上调。ChIP（染色质免疫沉淀）分析表明，在内质网胁迫下，ATF4、ATF3和C/EBPβ （CCAAT/增强子结合蛋白β）结合到ASNS近端启动子区域，该区域包括基因组序列NSRE（营养感应反应元件）-1和NSRE-2，这些基因组序列先前与UPR激活中的突变有关。与ASNS转录增加一致，ChIP分析还表明，UPR信号传导导致包括RNA Pol II（聚合酶II）在内的一般转录因子向ASNS启动子募集增加。在组织或细胞的氨基酸被剥夺后，ASNS基因也被AAR（氨基酸反应）途径激活。HepG2细胞的免疫印迹分析表明，与单独激活AAR和UPR通路相比，同时激活AAR和UPR通路并没有进一步增加ASNS或ATF4蛋白的丰度。此外，siRNA（小干扰RNA）介导的XBP1 （X-box结合蛋白1）、ATF6α或ATF6β表达的下调不影响ASNS转录，而ATF4的siRNA在UPR激活过程中抑制ASNS转录。综上所述，这些结果表明PERK/p-eIF2α（磷酸化真核起始因子2α）/ATF4信号级联是UPR中唯一负责内质网应激下ASNS转录诱导的臂。因此，ASNS NSRE-1和NSRE-2元件，以及ERSE （ER应激反应元件）- 1、ERSE- ii和mUPRE（哺乳动物UPR元件）都是哺乳动物UPR响应序列。
The UPR (unfolded protein response) pathway is comprised of three signalling cascades mediated by the ER (endoplasmic reticulum) stress sensor proteins PERK [PKR (double-stranded RNA-activated protein kinase)-like ER kinase], IRE1 (inositol-requiring kinase 1) and ATF6 (activating transcription factor 6). The present study shows that ASNS (asparagine synthetase) transcription activity was up-regulated in HepG2 cells treated with the UPR activators thapsigargin and tunicamycin. ChIP (chromatin immunoprecipitation) analysis demonstrated that during ER stress, ATF4, ATF3 and C/EBPβ (CCAAT/enhancer-binding protein β) bind to the ASNS proximal promoter region that includes the genomic sequences NSRE (nutrient-sensing response element)-1 and NSRE-2, previously implicated by mutagenesis in UPR activation. Consistent with increased ASNS transcription, ChIP analysis also demonstrated that UPR signalling resulted in enhanced recruitment of general transcription factors, including RNA Pol II (polymerase II), to the ASNS promoter. The ASNS gene is also activated by the AAR (amino acid response) pathway following amino acid deprivation of tissue or cells. Immunoblot analysis of HepG2 cells demonstrated that simultaneous activation of the AAR and UPR pathways did not further increase the ASNS or ATF4 protein abundance when compared to triggering either pathway alone. In addition, siRNA (small interfering RNA)-mediated knockdown of XBP1 (X-box binding protein 1), ATF6α or ATF6β expression did not affect ASNS transcription, whereas siRNA against ATF4 suppressed ASNS transcription during UPR activation. Collectively, these results indicate that the PERK/p-eIF2α (phosphorylated eukaryotic initiation factor 2α)/ATF4 signalling cascade is the only arm of the UPR that is responsible for ASNS transcriptional induction during ER stress. Consequently, the ASNS NSRE-1 and NSRE-2 elements, in addition to ERSE (ER stress response element)-I, ERSE-II and the mUPRE (mammalian UPR element), function as mammalian UPR responsive sequences.