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Regulation of Vascular Endothelial Growth Factor D by Orphan Receptors Hepatocyte Nuclear Factor-4 and Chicken Ovalbumin Upstream Promoter Transcription Factors 1 and 2

¹ Laboratory for Angiogenesis and Tumor Metastasis, Charité University Hospital, Berlin, Germany and ² Howard Hughes Medical Institute, La Jolla, California

Requests for reprints: Michael Höcker, Charité Universitätsmedizin Berlin, Campus Mitte, Schumannstraze 20/21, 10117 Berlin, Germany. Phone: 49-30-450-559709; Fax: 49-30-450-559989; E-mail: michael.hoecker{at}charite.de.

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 (HNF-4) 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 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. [Cancer Res 2008;68(2):457–66]