Mechanisms underlying TGF-β1-induced expression of VEGF and Flk-1 in mouse macrophages and their implications for angiogenesis

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-beta 1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia-inducible factor-1 alpha/beta (HIF-1 alpha/beta). To examine the transcriptional regulation of VEGF by TGF-beta 1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF-1 alpha/beta or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF-beta 1, whereas cotransfection of HIF-1 alpha/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-beta 1-induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin inummoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIIF-1 alpha/beta and Smad3/4, and EIA, an inhibitor of p300, inhibited it. TGF-beta 1 I also increased the expression of fetal liver kinase-1 (Flk-1), a major VEGF receptor, and TGF-beta 1 and VEGF stimulated pro-matrix metalloproteinase 9 (MMP-9) and active-MMP-9 expression, respectively. The results from the present study indicate that TGF-beta 1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP-9, and Flk-1.