Endoplasmic reticulum stress-induced hepatic stellate cell apoptosis through calcium-mediated JNK/P38 MAPK and Calpain/Caspase-12 pathways
Endoplasmic reticulum stress-induced hepatic stellate cell apoptosis through calcium-mediated JNK/P38 MAPK and Calpain/Caspase-12 pathways
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内质网应激通过钙介导的JNK/P38 MAPK和Calpain/Caspase-12途径诱导肝星状细胞凋亡
DOI：
10.1007/s11010-014-2073-8
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发表时间：
2014-09-01
影响因子：
4.3
通讯作者：
Li, Jun
中科院分区：
文献类型：
--
作者：
Huang, Yan;Li, Xiaohui;Li, Jun
近年来的研究认为，肝星状细胞（hepatic stellate cells，HSCs）凋亡的发生与内质网（endoplasmic reticulum，ER）内钙稳态的紊乱和错误折叠蛋白的积累有关，从而促进了肝纤维化的消退。然而，在HSC活化后由ER应激激活的信号转导事件并不完全清楚。在这项研究中，我们诱导内质网应激毒胡萝卜素（TG），并确定激活的钙蛋白酶和半胱天冬酶的裂解，通过分析蛋白水平和相应增加的细胞内钙水平和诱导的促凋亡转录因子CHOP。此外，JNK和p38 MAPK的磷酸化之后是执行者caspase-3的激活。正如预期的那样，使用细胞内钙螯合剂EGTA和BAPTA/AM防止细胞内钙水平的增加，可以显著抑制JNK和p38 MAPK的磷酸化，消除钙蛋白酶的激活，即半胱天冬酶-12，半胱天冬酶-9和半胱天冬酶-3，并为TG处理的活化HSC提供显著的保护。有趣的是，用p38 MAPK抑制剂SB 202190、JNK抑制剂SP 600125、泛半胱天冬酶抑制剂z-VAD-FMK或钙蛋白酶抑制剂calpeptin预处理显著减少细胞凋亡和半胱天冬酶-12和半胱天冬酶-3的切割。然而，z-VAD-FMK预处理未能减少钙蛋白酶的激活。SB 202190和SP 600125预处理也能降低CHOP的表达。重要的是，PDGF诱导的胶原Col 1 α1和α-平滑肌肌动蛋白（α-SMA）（HSC活化永久阶段的标志物）在TG处理的活化HSC中受到抑制。这些结果表明，内质网应激诱导的Calpain/Caspase-12活化和内质网释放的细胞内钙离子浓度增加诱导的JNK/p38 MAPK磷酸化是纤维化恢复的分子机制的新的信号通路。
Recent reports considered that it was the disturbance of calcium homeostasis and the accumulation of misfolded proteins in the endoplasmic reticulum (ER) that activated hepatic stellate cells (HSCs) apoptosis and promoted fibrosis resolution. However, the signal-transducing events that are activated by ER stress after HSCs activation were incompletely understood. In this study, we induced ER stress with thapsigargin (TG), and determined the activation of calpain and the cleavage of caspase by analyzing the protein levels and the correspondingly increased intracellular calcium levels and the induction of the proapoptotic transcription factor CHOP. Moreover, the phosphorylation of JNK and p38 MAPK were followed by the activation of the executioner caspases, caspase-3. As expected, preventing an increase in intracellular calcium levels using intracellular calcium chelators, EGTA, and BAPTA/AM, could substantially inhibit the phosphorylation of JNK and p38 MAPK, abolish the activation of calpains, namely caspase-12, caspase-9, and caspase-3, and provide significant protection for TG-treated activated HSCs. Interestingly, pretreatment with p38 MAPK inhibitor SB202190, JNK inhibitor SP600125, the pan-caspase inhibitor z-VAD-FMK, or calpain inhibitors calpeptin, significantly reduced the cell apoptosis and the cleavage of caspase-12 and caspase-3. However, pretreatment with z-VAD-FMK failed to reduce the activation of calpain. Additionally, pretreatment with SB202190 and SP600125 also decreased the expression of CHOP. Importantly, PDGF-induced collagen Col1α1 and α-smooth muscle actin (α-SMA), markers for the perpetuation phase of HSCs activation, were inhibited in TG-treated activated HSCs. These findings showed that the Calpain/Caspase-12 activation induced by ER stress and the JNK/p38 MAPK phosphorylation induced by the increase of intracellular calcium concentration releasing from ER are the novel signaling pathway underlying the molecular mechanism of fibrosis recovery.