MG132 induced apoptosis is associated with p53-independent induction of pro-apoptotic Noxa and transcriptional activity of β-catenin
MG132 induced apoptosis is associated with p53-independent induction of pro-apoptotic Noxa and transcriptional activity of β-catenin
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DOI：
10.1007/s10495-006-4990-9
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发表时间：
2006-04-01
期刊：
影响因子：
7.2
通讯作者：
Stott, NS
中科院分区：
文献类型：
--
作者：
Jüllig, M;Zhang, WV;Stott, NS
NOXA是BH3家族中唯一的促凋亡蛋白家族成员，在DNA损伤或生长因子剥夺等细胞应激反应中，核蛋白P53在转录水平上上调该蛋白的表达。NOXA能够与抗凋亡的Bcl2家族成员相互作用，导致细胞色素c释放到胞浆中，从而激活caspase，诱导细胞凋亡。在这里，我们证明了蛋白酶体抑制剂MG132可以快速诱导T/C28a和Saos2两个人类细胞株中的Noxa mRNA和蛋白。NOXA的诱导与MG132作用的前24小时代谢活跃的细胞数量显著减少以及caspase-3的逐渐激活有关，caspase-3是caspase依赖的细胞凋亡的标志。在用MG132处理细胞之前，将Noxa siRNA导入细胞，可以部分挽救细胞的表型，这表明NoxA的诱导具有功能意义。P53在T/C28a细胞系中不起作用，而在Saos2细胞中不存在，这表明NoxA的诱导是通过P53非依赖的机制实现的。Western blotting和共聚焦显微镜显示，在NOXA诱导时，两种细胞中的总β-连环蛋白都增加了，并且大部分β-连环蛋白存在于细胞核中。Tcf报告载体pTOPFLASH的转染证实，MG132处理可导致T/C28a和Saos2细胞中β-连环蛋白转录活性的早期增加。然而，尽管转录激活的β-连环蛋白在T/C28a细胞中的过度表达也通过非p53机制诱导细胞凋亡，但NOXA蛋白的水平没有变化，提示β-连环蛋白介导的信号转导和NOXA可能在MG132诱导的细胞凋亡中发挥独立的作用。综上所述，我们的结果表明，MG132通过P53非依赖的机制诱导促凋亡蛋白NoxA，该机制导致caspase依赖的细胞凋亡。这是第一个表明用MG132治疗可以诱导NOXA的报告。这项研究还为β-连环蛋白介导的信号和诱导细胞凋亡之间的联系提供了进一步的证据。
Noxa is a pro-apoptotic BH3-only member of the Bcl-2 family of proteins that is up-regulated at a transcriptional level by the nuclear protein p53 in response to cellular stresses such as DNA damage or growth factor deprivation. Noxa is able to interact with anti-apoptotic members of the Bcl-2 family and causes release of cytochrome c into the cytosol, leading to the activation of caspases and induction of apoptosis. Here we demonstrate that MG132, a proteasomal inhibitor, rapidly induces Noxa mRNA and protein in two human cell lines, T/C28a and Saos2. The induction of Noxa is associated with a significant reduction in the number of metabolically active cells over the first 24 h of exposure to MG132 and progressive activation of caspase-3, a hallmark of caspase-dependent apoptosis. Partial rescue of the phenotype is observed when cells are transfected with Noxa siRNA prior to treatment with MG132, indicating functional significance of the induction of Noxa. p53 has previously been shown to be non-functional in the T/C28a cell line and is absent by Western blotting in Saos2 cells, suggesting that the induction of Noxa is through a p53 independent mechanism. Western blotting and confocal microscopy showed that total beta-catenin protein is increased in both cell lines at the time of Noxa induction, with the bulk of the beta-catenin present in the nucleus. Transfection with the Tcf reporter vector pTOPFLASH confirms that treatment with MG132 leads to early increased transcriptional activity of beta-catenin in both T/C28a and Saos2 cells. However, although over-expression of transcriptionally active beta-catenin in T/C28a cells also induced apoptosis through a p53-independent mechanism, the levels of Noxa protein were unchanged, suggesting that beta-catenin mediated signaling and Noxa may play independent roles in MG132 induced apoptosis. In summary, our results demonstrate that MG132 induces the pro-apoptotic protein Noxa via a p53-independent mechanism that leads to caspase-dependent apoptosis. This is the first report showing that treatment with MG132 induces Noxa. This study also provides further evidence for a link between beta-catenin mediated signaling and the induction of apoptosis.