Alfonso-Jaume Maria Alejandra, Bergman Marina R, Mahimkar Rajeev, Cheng Sunfa, Jin Zhu Q, Karliner Joel S, Lovett David H
Dept. of Medicine, 111J, San Francisco Veterans Affairs Medical Center/Univ. of California San Francisco, 4150 Clement St., San Francisco, CA 94121, USA.
Am J Physiol Heart Circ Physiol. 2006 Oct;291(4):H1838-46. doi: 10.1152/ajpheart.00026.2006. Epub 2006 May 12.
Matrix metalloproteinase-2 (MMP-2) is a central component of the response to injury in the heart. During ischemia, MMP-2 influences ventricular performance and is a determinant of postinfarction remodeling. Elevation of MMP-2 during reperfusion after ischemia suggests that new protein is synthesized, but the molecular regulation of MMP-2 generation during ischemia-reperfusion (I/R) injury has not been studied. Using the MMP-2 promoter linked to a beta-galactosidase reporter in transgenic mice, we investigated the transcriptional regulation and cellular sources of MMP-2 in isolated, perfused mouse hearts subjected to acute global I/R injury. I/R injury induced a rapid activation of MMP-2 promoter activity with the appearance of beta-galactosidase antigen in cardiomyocytes, fibroblasts, and endothelial cells. Activation of intrinsic MMP-2 transcription and translation was confirmed by real-time PCR and quantitative Western blot analyses. MMP-2 transcription and translation were inhibited by perfusion with 1.0 mM hydroxyl radical scavenger N-(-2-mercaptopropionyl)-glycine. Nuclear extracts demonstrated increased abundance of two activator proteins-1 (AP-1) components JunB and FosB following I/R injury. Immunohistochemical staining localized JunB and FosB proteins to the nuclei of all three cardiac cell types following I/R injury, consistent with enhanced nuclear transport of these transcription factors. Chromatin immunoprecipitation (ChIP) of the AP-1 binding site in the intrinsic murine MMP-2 promoter yielded only JunB under control conditions, whereas ChIP following I/R injury recovered both JunB and FosB, consistent with a change in occupancy from JunB homodimers in controls to JunB/FosB heterodimers following I/R injury. We conclude that enhanced MMP-2 transcription and translation following I/R injury are mediated by induction, via oxidant stress, of discrete AP-1 transcription factor components.
基质金属蛋白酶-2（MMP-2）是心脏损伤反应的核心成分。在缺血期间，MMP-2影响心室功能，并且是梗死后重塑的一个决定因素。缺血后再灌注期间MMP-2的升高表明有新的蛋白质合成，但缺血-再灌注（I/R）损伤期间MMP-2生成的分子调控尚未得到研究。利用与β-半乳糖苷酶报告基因相连的MMP-2启动子，我们在遭受急性全心I/R损伤的离体灌注小鼠心脏中研究了MMP-2的转录调控和细胞来源。I/R损伤诱导MMP-2启动子活性迅速激活，同时在心肌细胞、成纤维细胞和内皮细胞中出现β-半乳糖苷酶抗原。通过实时PCR和定量蛋白质免疫印迹分析证实了内源性MMP-2转录和翻译的激活。用1.0 mM羟基自由基清除剂N-（-2-巯基丙酰基）-甘氨酸灌注可抑制MMP-2的转录和翻译。核提取物显示，I/R损伤后两种激活蛋白-1（AP-1）成分JunB和FosB的丰度增加。免疫组织化学染色显示，I/R损伤后JunB和FosB蛋白定位于所有三种心脏细胞类型的细胞核中，这与这些转录因子核转运增强一致。在对照条件下，对小鼠内源性MMP-2启动子中AP-1结合位点进行染色质免疫沉淀（ChIP）仅得到JunB，而I/R损伤后的ChIP同时回收了JunB和FosB，这与从对照中的JunB同二聚体到I/R损伤后的JunB/FosB异二聚体的占据变化一致。我们得出结论，I/R损伤后MMP-2转录和翻译增强是由氧化应激诱导离散的AP-1转录因子成分介导的。
