Abstract
TGF-beta induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF-beta1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia-inducible factor-1alpha/beta (HIF-1alpha/beta). To examine the transcriptional regulation of VEGF by TGF-beta1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF-1alpha/beta or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF-beta1, whereas cotransfection of HIF-1alpha/beta and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF-1 and two Smad-binding elements were critical for TGF-beta1-induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF-1alpha/beta and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF-beta1 also increased the expression of fetal liver kinase-1 (Flk-1), a major VEGF receptor, and TGF-beta1 and VEGF stimulated pro-matrix metalloproteinase 9 (MMP-9) and active-MMP-9 expression, respectively. The results from the present study indicate that TGF-beta1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP-9, and Flk-1.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cells, Cultured
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E1A-Associated p300 Protein / metabolism
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Enzyme-Linked Immunosorbent Assay / methods
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Gene Expression Profiling
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Hypoxia-Inducible Factor 1, alpha Subunit / metabolism
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Macrophages / drug effects
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Macrophages / immunology
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Matrix Metalloproteinase 9 / drug effects
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Matrix Metalloproteinase 9 / genetics
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Matrix Metalloproteinase 9 / metabolism
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Mice
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Mice, Inbred BALB C
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Neovascularization, Physiologic / drug effects
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Neovascularization, Physiologic / immunology*
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Promoter Regions, Genetic / genetics
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Protein Binding
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Reverse Transcriptase Polymerase Chain Reaction / methods
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Smad2 Protein / metabolism
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Smad3 Protein / metabolism
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Smad4 Protein / metabolism
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Structure-Activity Relationship
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Transcription, Genetic / drug effects
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Transcription, Genetic / genetics
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Transforming Growth Factor beta1 / pharmacology*
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Vascular Endothelial Growth Factor A / biosynthesis*
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Vascular Endothelial Growth Factor A / genetics*
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Vascular Endothelial Growth Factor Receptor-1 / genetics
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Vascular Endothelial Growth Factor Receptor-1 / metabolism
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Vascular Endothelial Growth Factor Receptor-2 / biosynthesis*
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Vascular Endothelial Growth Factor Receptor-2 / genetics*
Substances
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Hif1a protein, mouse
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Hypoxia-Inducible Factor 1, alpha Subunit
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Smad2 Protein
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Smad2 protein, mouse
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Smad3 Protein
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Smad3 protein, mouse
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Smad4 Protein
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Smad4 protein, mouse
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Transforming Growth Factor beta1
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Vascular Endothelial Growth Factor A
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E1A-Associated p300 Protein
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Ep300 protein, mouse
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Vascular Endothelial Growth Factor Receptor-1
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Vascular Endothelial Growth Factor Receptor-2
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Matrix Metalloproteinase 9