Transcriptional up-regulation of endothelial cell matrix metalloproteinase-2 in response to extracellular cues involves GATA-2.
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.
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D042783:Endothelial Cells / Q000201:enzymology* / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D050989:GATA2 Transcription Factor; D005780:Gelatin / Q000494:pharmacology; D017930:Genes, Reporter; D008156:Luciferases / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis* / Q000378:metabolism; D008833:Microcirculation / Q000378:metabolism; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation
D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; C494713:Gata2 protein, rat; D014157:Transcription Factors; D005780:Gelatin; D003094:Collagen; D008156:Luciferases; D020778:Matrix Metalloproteinase 2