Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium: a novel function beyond muscle contraction
Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium: a novel function beyond muscle contraction
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核心肌肌球蛋白轻链 2 调节心肌中 NADPH 氧化酶 2 的表达：肌肉收缩之外的新功能
DOI：
10.1007/s00395-015-0494-5
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发表时间：
2015-07-01
影响因子：
9.5
通讯作者：
Li, Qingjie
中科院分区：
文献类型：
--
作者：
Zhang, Yi-Shuai;Liu, Bin;Li, Qingjie
近年来的研究表明，缺血再灌注（IR）后，心肌组织中NADPH氧化酶2（NOX 2）的表达显著上调。然而，其潜在机制仍不清楚。本研究旨在确定核心肌肌球蛋白轻链2（MYL2），一个众所周知的肌球蛋白的调节亚基，作为一个转录因子，以磷酸化依赖的方式促进心肌IR后NOX 2的表达。我们检测了大鼠心肌IR（左冠状动脉主干结扎1 h，再灌注3 h）损伤模型中细胞核MYL2（p-MYL2）的磷酸化状态，结果显示IR损伤和预期的NOX2表达上调，伴随H2O2和细胞核p-MYL2水平升高;这些作用可通过抑制肌球蛋白轻链激酶（MLCK）而减弱。接下来，我们探讨了核p-MYL 2与H9c2细胞缺氧-复氧（HR）损伤模型中NOX 2表达的功能关系。与我们的体内研究结果一致，HR处理增加了细胞凋亡、NOX 2表达、核p-MYL2和H2O2水平，并且通过MLCK抑制或MYL2敲低来改善这些增加。最后，利用免疫共沉淀（Co-IP）、染色质免疫共沉淀（ChIP）、DNA pull-down和荧光素酶报告基因分析等分子生物学技术，研究了其分子机制。我们发现，核p-MYL2结合到NOX2基因启动子的共有序列AGCTCC，与RNA聚合酶II和转录因子IIB相互作用，形成转录前起始复合物，从而激活NOX2基因的转录。我们的研究结果表明，核MYL 2在IR损伤中起着重要作用，通过转录上调NOX 2的表达，以磷酸化依赖的方式增强氧化应激。
Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia-reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H2O2 and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H2O2 levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.