Transcriptional Up-regulation of Endothelial Cell Matrix Metalloproteinase-2 in Response to Extracellular Cues Involves GATA-2

Xiaoyan Han,Pamela J Boyd,Stephen Colgan,Joseph A Madri,Tara L Haas

doi:10.1074/jbc.m309482200

Xiaoyan Han, Pamela J Boyd + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m309482200

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Abstract

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.

Highlights

The matrix metalloproteinases (MMPs)1 consist of a family of zinc- and calcium-dependent endopeptidases that cleave specific subsets of extracellular matrix proteins, growth factors, and cell surface receptors [1,2]
Gel shift assays using double-stranded oligonucleotides corresponding to base pairs Ϫ1327 to Ϫ1293 of the Matrix metalloproteinase-2 (MMP-2) promoter showed two shifted complexes; the faster mobility complex was of similar intensity in nuclear extracts of cells in two-dimensional compared with three-dimensional cultures, whereas the slower mobility complex was greatly reduced in three-dimensional compared with two-dimensional nuclear extracts (Fig. 2D)
This study shows that the transcription factor, GATA-2, enhances the transcription of MMP-2 in microvascular endothelial cells cultured within a three-dimensional type I collagen matrix

Summary

Introduction

The matrix metalloproteinases (MMPs) consist of a family of zinc- and calcium-dependent endopeptidases that cleave specific subsets of extracellular matrix proteins, growth factors, and cell surface receptors [1,2]. The rate of MMP-2 protein production is controlled largely through alterations in mRNA transcription, proteolytic activity is regulated through secretion, cell surface binding, and activation of the latent enzyme [2]. Binding proteins within the RE1 domain are well characterized and include Y-box protein-1 (YB-1), p53, and AP-2 [10, 11], as well as the transcriptional suppressor, nm23-␤ [12] These transcription factors contribute greatly to the constitutive expression of MMP-2 in rat mesangial cells. Characterization of the promoter in rat endothelial cells identified an additional enhancer region (RE2) at base pairs Ϫ1435/Ϫ1375 [13]. We have shown that MMP-2 production in rat microvascular endothelial cells is sensitive to the extracellular matrix environment and is largely unresponsive to growth factor stimula-

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