A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers

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Abstract
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 24h 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 1β. 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 91bp region from −1433 to −1342bp, 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 −1362bp that included a potential activating protein 1 (AP-1)-binding sequence, C-1394CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the −1410 to −1362bp sequence. Electrophoretic mobility shift assays (EMSAs) using the −1410 to −1362bp 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 −1362bp 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.