Bergman Marina R, Cheng Sunfa, Honbo Norman, Piacentini Lucia, Karliner Joel S, Lovett David H
Cardiology Section, Department of Medicine, San Francisco Veterans Affairs Medical Center, University of California, 4150 Clement Street, San Francisco, CA 94121, USA.
Biochem J. 2003 Feb 1;369(Pt 3):485-96. doi: 10.1042/BJ20020707.
Enhanced synthesis of a specific matrix metalloproteinase, MMP-2, has been demonstrated in experimental models of ventricular failure and in cardiac extracts from patients with ischaemic cardiomyopathy. Cultured neonatal rat cardiac fibroblasts and myocytes were used to analyse the determinants of MMP-2 synthesis, including the effects of hypoxia. Culture of rat cardiac fibroblasts for 24 h in 1% oxygen enhanced MMP-2 synthesis by more than 5-fold and augmented the MMP-2 synthetic responses of these cells to endothelin-1, angiotensin II and interleukin 1beta. A series of MMP-2 promoter-luciferase constructs were used to map the specific enhancer element(s) that drive MMP-2 transcription in cardiac cells. Deletion studies mapped a region of potent transactivating function within the 91 bp region from -1433 to -1342 bp, the activity of which was increased by hypoxia. Oligonucleotides from this region were cloned in front of a heterologous simian-virus-40 (SV40) promoter and mapped the enhancer activity to a region between -1410 and -1362 bp that included a potential activating protein 1 (AP-1)-binding sequence, C(-1394)CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the -1410 to -1362 bp sequence. Electrophoretic mobility shift assays (EMSAs) using the -1410 to -1362 bp oligonucleotide and rat cardiac fibroblast nuclear extracts demonstrated specific nuclear-protein binding that was eliminated by cold competitor oligonucleotide, but not by the AP-1-mutated oligonucleotide. Antibody-supershift EMSAs of nuclear extracts from normoxic rat cardiac fibroblasts demonstrated Fra1 and JunB binding to the -1410 to -1362 bp oligonucleotide. Nuclear extracts isolated from hypoxic rat cardiac fibroblasts contained Fra1, JunB and also included FosB. Co-transfection of cardiac fibroblasts with Fra1-JunB and FosB-JunB expression plasmids led to significant increases in transcriptional activity. These studies demonstrate that a functional AP-1 site mediates MMP-2 transcription in cardiac cells through the binding of distinctive Fra1-JunB and FosB-JunB heterodimers. The synthesis of MMP-2 is widely considered, in contrast with many members of the MMP gene family, to be independent of the AP-1 transcriptional complex. This report is the first demonstration that defined members of the Fos and Jun transcription-factor families specifically regulate this gene under conditions relevant to critical pathophysiological processes.
在心室衰竭的实验模型以及缺血性心肌病患者的心脏提取物中，已证实一种特定的基质金属蛋白酶MMP - 2的合成增强。培养的新生大鼠心脏成纤维细胞和心肌细胞被用于分析MMP - 2合成的决定因素，包括缺氧的影响。将大鼠心脏成纤维细胞在1%氧气中培养24小时，可使MMP - 2合成增加5倍以上，并增强这些细胞对内皮素 - 1、血管紧张素II和白细胞介素1β的MMP - 2合成反应。一系列MMP - 2启动子 - 荧光素酶构建体被用于定位在心脏细胞中驱动MMP - 2转录的特定增强子元件。缺失研究确定了在 - 1433至 - 1342 bp的91 bp区域内有一个具有强大反式激活功能的区域，缺氧可增加其活性。来自该区域的寡核苷酸被克隆到异源猿猴病毒40（SV40）启动子之前，并将增强子活性定位到 - 1410至 - 1362 bp之间的一个区域，该区域包含一个潜在的激活蛋白1（AP - 1）结合序列C(-1394)CTGACCTCC。核心TGAC序列（加粗显示）的位点特异性诱变消除了 - 1410至 - 1362 bp序列内的反式激活活性。使用 - 1410至 - 1362 bp寡核苷酸和大鼠心脏成纤维细胞核提取物进行的电泳迁移率变动分析（EMSA）表明存在特异性的核蛋白结合，冷竞争寡核苷酸可消除这种结合，但AP - 1突变寡核苷酸不能。对常氧大鼠心脏成纤维细胞核提取物进行的抗体超迁移EMSA表明，Fra1和JunB与 - 1410至 - 1362 bp寡核苷酸结合。从缺氧大鼠心脏成纤维细胞中分离的核提取物含有Fra1、JunB，还包括FosB。将Fra1 - JunB和FosB - JunB表达质粒与心脏成纤维细胞共转染导致转录活性显著增加。这些研究表明，一个功能性的AP - 1位点通过独特的Fra1 - JunB和FosB - JunB异二聚体的结合介导心脏细胞中MMP - 2的转录。与MMP基因家族的许多成员相比，MMP - 2的合成被广泛认为独立于AP - 1转录复合物。本报告首次证明，Fos和Jun转录因子家族的特定成员在与关键病理生理过程相关的条件下特异性调节该基因。
