ZnT-1 protects HL-1 cells from simulated ischemia-reperfusion through activation of Ras-ERK signaling
ZnT-1 protects HL-1 cells from simulated ischemia-reperfusion through activation of Ras-ERK signaling
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DOI：
10.1007/s00109-011-0845-0
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发表时间：
2012-02-01
影响因子：
4.7
通讯作者：
Etzion, Yoram
中科院分区：
文献类型：
--
作者：
Beharier, Ofer;Dror, Shani;Etzion, Yoram
ERK信号通路的激活可能促进心肌缺血再灌注损伤的保护作用。ZNT-1是一种抵抗锌毒性的蛋白，它通过其C-末端结构域与Raf-1激酶相互作用，导致ERK下游激活。在本研究中，我们评估了ZNT-1对模拟I/R暴露的培养小鼠心肌细胞(HL-1细胞)的影响，通过乳酸脱氢酶(LDH)的释放和促凋亡caspase激活的染色来评估细胞损伤。在高表达ZNT-1的细胞中，高表达ZNT-1的细胞在I/R后磷酸化ERK水平显著增加，而下调ZNT-1则降低磷酸化ERK水平。用MEK抑制剂PD98059预处理细胞后，ZNT-1对细胞的保护作用消失。因此，缺失C末端结构域的截短型ZNT-1不能诱导ERK激活，也不能保护细胞免受I/R损伤。相反，C末端结构域的表达本身就足以诱导ERK激活和I/R保护。有趣的是，ZNT-1的C末端对锌的毒性没有保护作用。在分离的大鼠心脏中，全脑缺血损伤迅速增加内源性ZNT-1的水平。再灌流后，ZNT-1水平下降。我们的研究结果表明，ZNT-1可能通过与Raf-1激酶相互作用而在缺血心肌中发挥重要作用。
Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells over-expressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.