Investigation on the molecular mechanism of JNK-mediated apoptosis in cardiac myocytes

尽管氧化应激引起多种细胞类型的c-Jun氨基末端激酶(JNK)激活和细胞凋亡，但JNK通路如何与细胞凋亡途径相联系尚不清楚，本研究以成年大鼠心肌细胞作为氧化应激敏感的模型系统，研究了JNK介导的细胞凋亡的分子机制。氧化应激导致JNK活化、细胞色素c释放和细胞凋亡，没有新的蛋白质合成。氧化应激诱导的细胞凋亡可被显性负性SEK1介导的JNK通路抑制所消除，而JNK通路的激活足以引起细胞凋亡。抑制线粒体凋亡途径中的顶端caspase-9可抑制氧化应激诱导的细胞凋亡，而抑制caspase-8则不起作用，表明JNK途径和线粒体凋亡机制在氧化应激诱导的细胞凋亡中起中心作用。JNK和Seki都定位在线粒体上，氧化应激激活了JNK。此外，在无细胞实验中，激活的JNK使几种线粒体蛋白磷酸化，并导致细胞色素c等致凋亡因子从分离的线粒体中释放出来。这些发现表明，JNK途径是线粒体死亡机制的直接激活者，而不是其他细胞成分，并提供了从氧化应激到线粒体凋亡机制的分子联系。此外，JNK还激活血管平滑肌细胞和巨噬细胞中的基质金属蛋白酶，从而调节心血管系统的组织重构。

Although oxidative stress causes activation of c-Jun N-terminal kinase (JNK) and apoptosis in many cell types, how the JNK pathway is connected to the apoptosis pathway is unclear, The molecular mechanism of JNK-mediated apoptosis was investigated in adult rat cardiac myocytes in culture as a model system that is sensitive to oxidative stress. Oxidative stress caused JNK activation, cytochrome c release and apoptosis without new protein synthesis. Oxidative stress-induced apoptosis was abrogated by dominant negative SEK1-mediated inhibition of JNK pathway, whereas activation of JNK pathway by constitutively active SEKI was sufficient to cause apoptosis. Inhibition of caspase-9, an apical caspase in mitochondrial apoptosis pathway, suppressed oxidative stress-induced apoptosis, whereas inhibition of caspase-8 had no effect, indicating that both JNK pathway and mitochondrial apoptosis machinery are central to oxidative stress-induced apoptosis. Both JNK and SEKI localized on mitochondria where JNK was activated by oxidative stress. Furthermore, active JNK phosphorylated several mitochondrial proteins and caused the release of apoptogenic factors such as cytochrome c from isolated mitochondria in a cell-free assay. These findings indicate that the JNK pathway is a direct activator of mitochondrial death machinery without other cellular components and provide a molecular linkage from oxidative stress to the mitochondrial apoptosis machinery. In addition, JNK activates matrix metalloproteinases in vascular smooth muscle cells and macrophages, which regulates tissue remodeling of cardiovascular system