Campos Gisela, Schmidt-Heck Wolfgang, Ghallab Ahmed, Rochlitz Katharina, Pütter Larissa, Medinas Danilo B, Hetz Claudio, Widera Agata, Cadenas Cristina, Begher-Tibbe Brigitte, Reif Raymond, Günther Georgia, Sachinidis Agapios, Hengstler Jan G, Godoy Patricio
IfADo-Leibniz Research Centre for Working Environment and Human Factors, Technical University Dortmund, Ardeystrasse 67, 44139, Dortmund, Germany.
Arch Toxicol. 2014 Jun;88(6):1267-80. doi: 10.1007/s00204-014-1240-8. Epub 2014 Apr 20.
Since xenobiotics enter the organism via the liver, hepatocytes must cope with numerous perturbations, including modifications of proteins leading to endoplasmic reticulum stress (ER-stress). This triggers a signaling pathway termed unfolded protein response (UPR) that aims to restore homeostasis or to eliminate disturbed hepatocytes by apoptosis. In the present study, we used the well-established CCl4 hepatotoxicity model in mice to address the questions whether CCl4 induces ER-stress and, if so, whether the well-known ER-stress effector CHOP is responsible for CCl4-induced apoptosis. For this purpose, we treated mice with a high dose of CCl4 injected i.p. and followed gene expression profile over time using Affymetrix gene array analysis. This time resolved gene expression analysis allowed the identification of gene clusters with overrepresented binding sites for the three most important ER-stress induced transcription factors, CHOP, XBP1 and ATF4. Such result was confirmed by the demonstration of CCl4-induced XBP1 splicing, upregulation of CHOP at mRNA and protein levels, and translocation of CHOP to the nucleus. Two observations indicated that CHOP may be responsible for CCl4-induced cell death: (1) Nuclear translocation of CHOP was exclusively observed in the pericentral fraction of hepatocytes that deteriorate in response to CCl4 and (2) CHOP-regulated genes with previously reported pro-apoptotic function such as GADD34, TRB3 and ERO1L were induced in the pericentral zone as well. Therefore, we compared CCl4 induced hepatotoxicity in CHOP knockout versus wild-type mice. Surprisingly, genetic depletion of CHOP did not afford protection against CCl4-induced damage as evidenced by serum GOT and GPT as well as quantification of dead tissue areas. The negative result was obtained at several time points (8, 24 and 72 h) and different CCl4 doses (1.6 and 0.132 g/kg). Overall, our results demonstrate that all branches of the UPR are activated in mouse liver upon CCl4 treatment. However, CHOP does not play a critical role in CCl4-induced cell death and cannot be considered as a biomarker strictly linked to hepatotoxicity. The role of alternative UPR effectors such as XBP1 remains to be investigated.
由于外源性物质通过肝脏进入机体，肝细胞必须应对众多干扰，包括导致内质网应激（ER应激）的蛋白质修饰。这会触发一条称为未折叠蛋白反应（UPR）的信号通路，其目的是恢复内环境稳态或通过凋亡消除受干扰的肝细胞。在本研究中，我们使用了成熟的小鼠四氯化碳肝毒性模型来解决以下问题：四氯化碳是否诱导ER应激，如果是，著名的ER应激效应因子CHOP是否负责四氯化碳诱导的凋亡。为此，我们给小鼠腹腔注射高剂量四氯化碳，并使用Affymetrix基因芯片分析随时间跟踪基因表达谱。这种时间分辨基因表达分析使得能够鉴定出具有三个最重要的ER应激诱导转录因子CHOP、XBP1和ATF4的结合位点过度富集的基因簇。四氯化碳诱导的XBP1剪接、CHOP在mRNA和蛋白质水平的上调以及CHOP向细胞核的转位证实了这一结果。两项观察结果表明CHOP可能负责四氯化碳诱导的细胞死亡：（1）仅在因四氯化碳而受损的肝小叶中央区肝细胞中观察到CHOP的核转位，以及（2）先前报道具有促凋亡功能的CHOP调节基因如GADD34、TRB3和ERO1L也在肝小叶中央区被诱导。因此，我们比较了CHOP基因敲除小鼠和野生型小鼠中四氯化碳诱导的肝毒性。令人惊讶的是，血清谷草转氨酶和谷丙转氨酶以及死亡组织面积的定量结果表明，CHOP的基因缺失并不能提供对四氯化碳诱导损伤的保护作用。在几个时间点（8、24和72小时）以及不同的四氯化碳剂量（1.6和0.132 g/kg）下均得到了阴性结果。总体而言，我们的结果表明，四氯化碳处理后小鼠肝脏中UPR的所有分支均被激活。然而，CHOP在四氯化碳诱导的细胞死亡中不发挥关键作用，不能被视为与肝毒性严格相关的生物标志物。替代的UPR效应因子如XBP1的作用仍有待研究。
