Intronic regulation of matrix metalloproteinase-2 revealed by in vivo transcriptional analysis in ischemia

Abstract

Matrix metalloproteinase-2 (MMP-2) plays an essential role in angiogenesis and arteriogenesis, two processes critical to restoration of tissue perfusion after ischemia. MMP-2 expression is increased in tissue ischemia, but the responsible mechanisms remain unknown. We studied the transcriptional activation of the MMP-2 gene in a model of hindlimb ischemia by using various MMP-2-lacZ reporter mice and chromatin immunoprecipitation. MMP-2 activity and mRNA were increased after hindlimb ischemia. Mice with targeted deletion of MMP-2 had impaired restoration of perfusion and a high incidence of limb gangrene, indicating that MMP-2 plays a critical role in ischemia-induced revascularization. Ischemia induced the expression and binding of c-Fos, c-Jun, JunB, FosB, and Fra2 to a noncanonical activating protein-1 (AP-1) site present in the MMP-2 promoter and decreased binding of the transcriptional repressor JunD. Ischemia also activated the expression and binding of p53 to an adjacent enhancer site (RE-1) and increased expression and binding of nuclear factor of activated T-cells-c2 to consensus sequences within the first intron. Deletion of either the 5 AP-1͞ RE-1 region of the promoter or substitution of the first intron abolished ischemia-induced MMP-2 transcription in vivo. Thus, AP-1 transcription factors and intronic activation by nuclear factor of activated T-cells-c2 act in concert to drive ischemia-induced MMP-2 transcription. These findings define a critical role for MMP-2 in ischemia-induced revascularization and identify both previously uncharacterized regulatory elements within the MMP-2 gene and the cognate transcription factors required for MMP-2 activation in vivo after tissue ischemia.angiogenesis ͉ gelatinase ͉ gene expression ͉ skeletal muscle ͉ transcription factor T issue ischemia secondary to arterial ischemia remains a major source of morbidity and mortality. Despite their importance, the cellular and molecular mechanisms that drive transcription of the genes essential for reversal of ischemia in vivo remain largely undefined. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes important in tissue remodeling and repair. MMP-2 has been implicated in the collateral arterial enlargement that occurs in response to tissue ischemia (1), and its expression is essential for retinal and tumor-induced angiogenesis (2, 3). MMP-2 (gelatinase A) can cleave most extracellular matrix proteins, including collagen, and can proteolytically activate other proenzymes, such as MMP-9. The lack of MMP-14 (membrane-type 1-MMP), the cell surface protease that cleaves proMMP-2 to its active form, abolishes postnatal angiogenesis (4), and targeted deletion of MMP-9 impairs revascularization after hindlimb ischemia (5). Experimental hindlimb ischemia increases expression of MMP-2, MMP-9, and MMP-14 (6). Despite the potential importance of MMP-2 in ischemia-induced angiogenesis and arteriogenesis, the role of MMP-2 in this process and the transcriptional regulatory mechanisms that regulate MMP-2 in tissue is...

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Myocardial Matrix Remodeling and the Matrix Metalloproteinases: Influence on Cardiac Form and Function

“…In other studies, the full-length promoter region for MMP-2 or MMP-9 fused to a reporter has been permanently ligated into the murine system (4,220,286). With the use of this approach, MMP transcriptional activity can be quantified as well as localized.…”

“…The full-length human MMP-2 or MMP-9 promoter was ligated to the ␤-galactosidase reporter and fused into mice (4,220,286). This approach provides for spatial localization of MMP-2 and MMP-9 promoter activity.…”

Connective Tissue Growth Factor Regulates Retinal Neovascularization through p53 Protein-dependent Transactivation of the Matrix Metalloproteinase (MMP)-2 Gene

“…Ischemia induced the expression and binding of c-Fos, c-Jun, JunB, FosB, and Fra2 to a noncanonical AP-1 site present in the MMP-2 promoter and decreased binding of the transcriptional repressor JunD. In a model of hind limb ischemia, the MMP-2 promoter was also enhanced by increased expression and binding of nuclear factor of activated T-cells-c2 (NFATc2) to consensus sequences within the first intron (67). In this model, deletion of either the 5Ј regulatory element (RE) region of the promoter or substitution of the first intron abolished ischemia-induced MMP-2 transcription in vivo.…”