Abstract
Vascular endothelial growth factor D has recently been linked to the control of lymphangiogenesis and lymphatic metastasis. The molecular determinants regulating vegf-D gene transcription, however, have not yet been identified. After isolation of 2 kb of 5'-flanking DNA of the human vegf-D gene, we identified a novel, atypical direct repeat (DR) element consisting of a consensus half-site (AGGTCA) at -125/-119 and a degenerated DR half-site (ATGTTA) at -99/-94 as sufficient and necessary for vegf-D transcription. The vegf-D DR element is bound and activated by the orphan receptors hepatocyte nuclear factor 4 alpha (HNF-4 alpha) and chicken ovalbumin upstream promoter transcription factor (COUP-TF)-1/COUP-TF2. Additionally, chromatin immunoprecipitation assays identified transcriptional coactivators cyclic AMP-responsive element binding protein-binding protein and glucocorticoid receptor interacting protein 1 at the vegf-D DR element and functional assays confirmed their stimulatory effect on the vegf-D promoter. Histone deacetylase inhibition by trichostatin A led to accumulation of acetylated histones H3/H4 at the vegf-D promoter, up-regulation of vegf-D mRNA levels, and transactivation of vegf-D promoter reporter gene constructs in cancer cell lines. This study for the first time describes the molecular determinants in cis and trans controlling vegf-D gene transcription and identifies interaction of HNF-4 alpha and COUP-TF1/COUP-TF2 with a proximal, atypical DR element as indispensable for vegf-D transcription. Moreover, our findings suggest that epigenetic control of histone acetylation represents an important determinant of vegf-D gene expression in cancer cells. These results provide novel insights into the molecular machinery controlling vegf-D gene expression and may add to a better understanding of the regulation of lymphangiogenesis in vascular development and cancer.
Highlights
Angiogenesis, the formation of new blood vessels from endothelial precursors, is a prerequisite for growth and dissemination of solid malignancies [1], and the vascular endothelial growth factor (VEGF) superfamily of endothelial growth factors hasNote: Supplementary data for this article are available at Cancer Research Online.Despite the accumulating evidence supporting the importance of VEGF-C and VEGF-D for lymphangiogenesis and cancer spread, the current knowledge on molecular determinants and regulating pathways is very limited
Shortening of the vegfD 5¶ sequence by additional 40 bp [construct vegf-D(À83/+110)] abolished vegf-D transcription (Fig. 2A). Because these results indicated that vegf-D À141 to À83 contains essential regulatory elements, this sequence was used as a radiolabeled probe in Electrophoretic mobility shift assays (EMSA)
Showing unaffected binding of complex II or complex I, the activity conferred by vegf-D À135/À109 and vegf-D À109/À81 in pT81 was drastically reduced (Fig. 2D), suggesting that enhancer elements exist within the À138/À79 region that interact in a cooperative fashion
Summary
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Despite the accumulating evidence supporting the importance of VEGF-C and VEGF-D for lymphangiogenesis and cancer spread, the current knowledge on molecular determinants and regulating pathways is very limited. Resistant to various extracellular stimulators, vegf-D mRNA levels were found to be stimulated by a cell-cell contact–triggered mechanism involving cadherin-11 [8]. It was shown that a h-catenin– dependent mechanism can down-regulate vegf-D expression by influencing mRNA stability [11]. Because the human vegf-D cDNA was initially isolated using an activator protein (AP-1) overexpression strategy [12], it was suggested that AP-1 may be directly involved in regulation of vegf-D gene expression. Because the transcription factors and regulatory promoter elements controlling vegf-D gene expression have not yet been identified, the role of AP1 in regulation of VEGF-D, remains hypothetical
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