Critical role of c-Jun N-terminal protein kinase activation in troglitazone-induced apoptosis of human HepG2 hepatoma cells
Critical role of c-Jun N-terminal protein kinase activation in troglitazone-induced apoptosis of human HepG2 hepatoma cells
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DOI：
10.1124/mol.63.2.401
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发表时间：
2003-02-01
影响因子：
3.6
通讯作者：
Song, BJ
中科院分区：
文献类型：
--
作者：
Bae, MA;Song, BJ
过氧化物酶体增殖物激活受体激动剂曲格列酮（TRO）用于治疗非胰岛素依赖型糖尿病，直到因其严重的肝毒性而从市场上撤下。然而，其肝毒性的机制仍然知之甚少。在这项研究中，我们研究了TRO是否通过改变与人类肝癌细胞的细胞损伤和存活相关的信号通路而导致细胞死亡。我们的数据显示，TRO引起HepG 2和Chang肝人肝癌细胞的时间和浓度依赖性凋亡，DNA片段化和Hoechst 33342染色证明。相反，50或100 μ M罗格列酮，TRO的结构类似物，没有引起这些肝癌细胞凋亡。TRO激活c-Jun N-末端蛋白激酶（JNK）和p38激酶约5倍之间的0.5和8小时，然后在16小时在HepG 2细胞中恢复到对照水平。相反，TRO未能激活细胞外信号调节激酶。此外，TRO增加促凋亡蛋白，坏，Bax的水平，释放细胞色素c，并在时间依赖性的方式裂解的投标。抗凋亡Bcl-2蛋白水平在TRO处理的肝癌细胞中降低。用选择性JNK抑制剂蒽[1，9-cd]吡唑-6（28）-酮（SP 600125）预处理肝癌细胞可显著降低TRO诱导的细胞死亡率，而p38激酶抑制剂4-（4-氟苯基）-2-（4-甲基亚磺酰基苯基）-5-（4-吡啶基）-1H-咪唑（SB 203580）对细胞凋亡几乎没有影响。用SP 600125预处理也阻止JNK活化和c-Jun磷酸化。此外，罗格列酮对肝癌细胞的毒性不如TRO，它不刺激JNK活性。转染显性失活突变体JNK-KR（Lys->Arg）或SEK 1-KR（Lys->Arg）（JNK的直接上游激酶）的cDNA显著降低TRO诱导的JNK激活和细胞死亡率。此外，SP 600125预处理有效地阻止了TRO介导的Bad、Bax、BID切割和细胞色素c释放的变化。这些数据强烈表明，肝毒性TRO通过激活JNK依赖性细胞死亡途径引起凋亡，伴随着增加Bid裂解和促凋亡蛋白的升高。
The peroxisome proliferator-activated receptor agonist troglitazone (TRO) was used for treatment of non-insulin-dependent diabetes until its removal from the market because of its severe hepatotoxicity. However, the mechanism for its hepatotoxicity is still poorly understood. In this study, we investigated whether TRO caused cell death by altering signaling pathways associated with cell damage and survival in human hepatoma cells. Our data reveal that TRO caused time- and concentration-dependent apoptosis of HepG2 and Chang liver human hepatoma cells, as evidenced by DNA fragmentation and staining with Hoechst 33342. In contrast, 50 or 100 muM rosiglitazone, a structural analog of TRO, did not cause apoptosis in these hepatoma cells. TRO activated both c-Jun N-terminal protein kinase (JNK) and p38 kinase about 5-fold between 0.5 and 8 h before they returned to control levels at 16 h in HepG2 cells. In contrast, TRO failed to activate the extracellular signal-regulated kinase. Furthermore, TRO increased the levels of proapoptotic proteins, Bad, Bax, release of cytochrome c, and cleavage of Bid in a time-dependent manner. The antiapoptotic Bcl-2 protein level decreased in hepatoma cells treated with TRO. Pretreatment of hepatoma cells with a selective JNK inhibitor, anthra[1,9-cd]pyrazol-6(28)-one (SP600125), significantly reduced the rate of TRO-induced cell death, whereas 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), an inhibitor of p38 kinase, had little effect on apoptosis. Pretreatment with SP600125 also prevented JNK activation and c-Jun phosphorylation. In addition, rosiglitazone, which is not as toxic to hepatoma cells as TRO, did not stimulate JNK activity. Transfection of cDNA for the dominant-negative mutant JNK-KR (Lys-->Arg) or SEK1-KR (Lys-->Arg), an immediate upstream kinase of JNK, significantly reduced TRO-induced JNK activation and cell death rate. Furthermore, SP600125 pretreatment effectively prevented the TRO-mediated changes in Bad, Bax, Bid cleavage, and cytochrome c release. These data strongly suggest that hepatotoxic TRO causes apoptosis by activating the JNK-dependent cell death pathway accompanied by increased Bid cleavage and elevation of proapoptotic proteins.