Han Xiaoyan, Boyd Pamela J, Colgan Stephen, Madri Joseph A, Haas Tara L
School of Kinesiology and Health Sciences, York University, Toronto, Ontario M3J 1P3, Canada.
J Biol Chem. 2003 Nov 28;278(48):47785-91. doi: 10.1074/jbc.M309482200. Epub 2003 Sep 25.
Matrix metalloproteinase-2 (MMP-2) plays a critical role in endothelial cells during the processes of angiogenesis and vascular remodeling. Endothelial cell production of MMP-2 is greatly enhanced when cells are cultured within a three-dimensional type I collagen matrix coinciding with the increased invasive and migratory phenotype of the cells. To define the transcriptional regulation of MMP-2 in rat microvascular endothelial cells, we performed promoter-reporter assays with a series of promoter truncations. Activity of the full promoter was significantly greater in cells cultured within three-dimensional type I collagen compared with cells cultured as a monolayer (two-dimensional) on type I collagen. Truncation of the region encompassing base pairs -1562 to -1375 (relative to the start codon) of the MMP-2 promoter resulted in loss of this differential activity of the MMP-2 promoter. Analysis of this region indicated two putative GATA-2 binding domains between -1437 and -1387. Southwestern blot analysis and electrophoretic mobility shift assays confirmed the binding of GATA-2 to this region of the MMP-2 promoter. Overexpression of GATA-2 in COS-7 cells significantly increased the activity of the full-length MMP-2 promoter-luciferase construct. Endothelial cells expressed greater levels of GATA-2 protein in three-dimensional compared with two-dimensional cultures, and activity of the -1437/-1387 region of the MMP-2 promoter was significantly greater in three-dimensional cultured endothelial cells. Together, these results indicate GATA-2 regulation of the MMP-2 promoter in endothelial cells and that the GATA-2 binding domain is sufficient to drive increased activity of the MMP-2 promoter in response to an extracellular matrix stimulus.
基质金属蛋白酶-2（MMP-2）在血管生成和血管重塑过程中对内皮细胞起着关键作用。当细胞在三维I型胶原基质中培养时，MMP-2的内皮细胞产生会大大增强，这与细胞侵袭性和迁移表型的增加相一致。为了确定大鼠微血管内皮细胞中MMP-2的转录调控，我们用一系列启动子截短片段进行了启动子-报告基因分析。与在I型胶原上单层（二维）培养的细胞相比，在三维I型胶原中培养的细胞中，全长启动子的活性显著更高。MMP-2启动子中包含碱基对-1562至-1375（相对于起始密码子）的区域被截短后，导致MMP-2启动子这种差异活性丧失。对该区域的分析表明，在-1437和-1387之间有两个假定的GATA-2结合结构域。蛋白质印迹分析和电泳迁移率变动分析证实了GATA-2与MMP-2启动子的该区域结合。在COS-7细胞中过表达GATA-2显著增加了全长MMP-2启动子-荧光素酶构建体的活性。与二维培养相比，内皮细胞在三维培养中表达更高水平的GATA-2蛋白，并且在三维培养的内皮细胞中，MMP-2启动子的-1437 / -1387区域的活性显著更高。总之，这些结果表明GATA-2在内皮细胞中对MMP-2启动子有调控作用，并且GATA-2结合结构域足以驱动MMP-2启动子活性增加以响应细胞外基质刺激。
