Oppositional Regulation of Noxa by JNK1 and JNK2 during Apoptosis Induced by Proteasomal Inhibitors

Proteasome inhibitors (PIs) potently induce apoptosis in a variety of tumor cells, but the underlying mechanisms are not fully elucidated. Comparing PI-induced apoptosis susceptibilities of various mouse embryonic fibroblast (MEF) lines differing in their c-jun N-terminal kinase (JNK) 1 and 2 status, we show that several hallmarks of apoptosis were most rapidly detectable in JNK2−/− cells, whereas they appeared only delayed and severely reduced in their intensities in cells expressing JNK2. Consistent with our finding that PI-induced apoptosis requires de novo protein synthesis, the proteasomal inhibitor MG-132 induced expression of the BH3-only protein Noxa at the transcriptional level in a JNK1-dependent, but JNK2-opposing manner. As the knockdown of Noxa blocked only the rapid PI-induced apoptosis of JNK2−/− cells, but not the delayed death occurring in JNK1−/− and JNK1+/+ cells, our data uncover a novel PI-induced apoptosis pathway that is regulated by the JNK1/2-dependent expression of Noxa. Furthermore, several transcription factors known to modulate Noxa expression including ATF3, ATF4, c-Jun, c-Myc, HIF1α, and p53 were found upregulated following MG-132 exposure. From those, only knockdown of c-Myc rescued JNK2−/− cells from PI-induced apoptosis, however, without affecting expression of Noxa. Together, our data not only show that a rapid execution of PI-induced apoptosis requires JNK1 for upregulation of Noxa via an as yet unknown transcription factor, but also that JNK2 controls this event in an oppositional manner.

蛋白酶体抑制剂（Proteasome inhibitors, PIs）可强效诱导多种肿瘤细胞发生细胞凋亡，但其具体分子机制尚未完全阐明。本研究通过对比不同c-Jun氨基末端激酶（c-jun N-terminal kinase, JNK）1和2基因状态的小鼠胚胎成纤维细胞（mouse embryonic fibroblast, MEF）系经PI处理后的凋亡易感性，发现JNK2基因敲除（JNK2−/−）细胞可最快检测到多项细胞凋亡标志性事件；而在表达JNK2的细胞中，这些事件仅延迟出现，且信号强度显著降低。本研究此前发现PI诱导的细胞凋亡依赖于从头蛋白质合成，进一步实验显示，蛋白酶体抑制剂MG-132可通过JNK1依赖、JNK2拮抗的方式，在转录水平上调BH3-only蛋白Noxa（BH3-only protein Noxa）的表达。基因敲低Noxa仅能阻断JNK2−/−细胞中PI诱导的快速凋亡，却无法抑制JNK1基因敲除（JNK1−/−）及JNK1野生型（JNK1+/+）细胞发生的延迟性死亡，由此我们揭示了一条全新的PI诱导细胞凋亡通路：该通路由JNK1/2依赖的Noxa表达调控。此外，MG-132处理后，多种已知可调控Noxa表达的转录因子（包括ATF3、ATF4、c-Jun、c-Myc、HIF1α及p53）的表达均出现上调。在这些转录因子中，仅敲低c-Myc可使JNK2−/−细胞免受PI诱导的凋亡，但该过程并不会影响Noxa的表达。综上，本研究不仅证实PI诱导的快速细胞凋亡需要JNK1通过尚未明确的转录因子上调Noxa的表达，同时也揭示了JNK2以拮抗方式调控这一过程的全新机制。