Nuclear translocation of MLKL enhances necroptosis by a RIP1/RIP3-independent mechanism in H9c2 cardiomyoblasts.
Nuclear translocation of MLKL enhances necroptosis by a RIP1/RIP3-independent mechanism in H9c2 cardiomyoblasts.
复制标题
MLKL 的核转位通过 RIP1/RIP3 独立机制增强 H9c2 心肌细胞中的坏死性凋亡。
DOI：
10.1016/j.jphs.2022.12.009
复制
发表时间：
2023
影响因子：
3.5
通讯作者：
Miura T.
中科院分区：
文献类型：
--
作者：
Ino S;Yano T;Kuno A;Tanno M;Kouzu H;Sato T;Yamashita T;Ohwada W;Osanami A;Ogawa T;Toda Y;Shimizu M;Miura T.
越来越多的证据表明心肌细胞坏死与心血管疾病有关。坏死下垂中质膜的致命破坏是由混合谱系激酶结构域样（MLKL）的低聚物诱导的，该低聚物从细胞质溶胶转移到膜上。然而，MLKL的细胞质-核穿梭所起的作用尚不清楚。在这里，我们验证了MLKL向细胞核的易位促进心肌细胞坏死的假设。TNF-α和zVAD联合激活典型的坏死坏死信号通路（TNF/zVAD）以rip1活性依赖的方式增加大鼠成心肌细胞系H9c2细胞的核MLKL水平。通过定点诱变，我们在MLKL中发现了一个核输出信号序列，并制备了其突变体（MLKL- l280 /283/ 284a），但没有找到核输入信号。MLKL-L280/283/284A定位于细胞质和细胞核。MLKL-L280/283/284A的表达可诱导坏死细胞死亡，GppNHp（一种ran介导的核输入抑制剂）可减轻这种死亡，但不能抑制RIP1活性或敲低RIP3的表达。GppNHp部分抑制TNF/zVAD诱导的H9c2细胞死亡。这些结果表明，通过RIP1介导的坏死信号转移到细胞核的MLKL通过RIP1-/ rip3独立的机制增强心肌细胞的坏死。
Accumulating evidence suggests that necroptosis of cardiomyocytes contributes to cardiovascular diseases. Lethal disruption of the plasma membrane in necroptosis is induced by oligomers of mixed lineage kinase domain-like (MLKL) that is translocated to the membrane from the cytosol. However, the role played by cytoplasmic-nuclear shuttling of MLKL is unclear. Here, we tested the hypothesis that translocation of MLKL to the nucleus promotes the necroptosis of cardiomyocytes. Activation of the canonical necroptotic signaling pathway by a combination of TNF-α and zVAD (TNF/zVAD) increased nuclear MLKL levels in a RIP1-activity-dependent manner in H9c2 cells, a rat cardiomyoblast cell line. By use of site-directed mutagenesis, we found a nuclear export signal sequence in MLKL and prepared its mutant (MLKL-L280/283/284A), though a search for a nuclear import signal was unsuccessful. MLKL-L280/283/284A localized to both the cytosol and the nucleus. Expression of MLKL-L280/283/284A induced necroptotic cell death, which was attenuated by GppNHp, an inhibitor of Ran-mediated nuclear import, but not by inhibition of RIP1 activity or knockdown of RIP3 expression. GppNHp partly suppressed H9c2 cell death induced by TNF/zVAD treatment. These results suggest that MLKL that is translocated to the nucleus via RIP1-mediated necroptotic signaling enhances the necroptosis of cardiomyocytes through a RIP1-/RIP3-independent mechanism.