Aims: Vascular endothelial growth factor D (VEGF-D) is a ligand for VEGF receptor 3 (VEGFR–3/flt4) and has been implicated in lymphatic development and metastases. VEGF-D expression correlates with lymphatic tumor spread and poor disease outcome; our own
studies identified VEGF-D as novel independent prognostic marker in gastric cancer.
Despite this clinical importance, the molecular mechanisms controlling VEGF-D are
largely unknown. Aim: Detailed characterization of the mechanisms controlling VEGF-D transcription as a basis for development of new intervention strategies influencing
lymphangiogenesis and lymphatic metastases. Methods: After initial localization of the VEGF-D minimal promoter between –135/–85, regulatory promoter elements were identified by
3’/5’ deletions, site-directed mutagenesis and element transfer studies. Nuclear proteins
and transcriptional co-factors controlling VEGF-D transcription were identified by EMSA and chromatin immunoprecipitation (ChIP) assays,
and confirmed by ectopic expression of wild type and dominant-negative factor mutants.
The role of acetylation for VEGF-D transcription was determined by application of HDAC inhibitor trichostatin (TSA).
Results: A novel, atypical direct repeat (DR) element located within the –138/–85 region was identified as sufficient and necessary
for VEGF-D gene transcription. This DR element comprises a 5’ consensus DR half-site (AGGTCA),
a 25 bp spacer and a 3’ degenerated DR sequence, and is controlled through an interplay
of the orphan receptors HNF–4α and COUP-TF1/2. Moreover, the transcriptional co-factors
Grip–1 and CBP were identified to cooperate with HNF–4α and COUP-TF1/2 in regulation
of VEGF-D transcription. Modification of acetylation resulted in potent and time-dependent
upregulation of VEGF-D mRNA levels and promoter activity. Conclusions: Our studies for the first time describe the molecular mechanisms controlling VEGF-D
gene transcription and show that the molecular machinery regulating the VEGF-D gene clearly differs from the mechanisms regulating other VEGF genes. Moreover, we
demonstrate that promoter acetylation is an important determinant of VEGF-D transcription and thereby may influence genetic programs underlying lymphatic proliferation
and metastasis
Schlüsselwörter
Vascular endothelial growth factor D - acetylation - direct repeat element - lymphatic
differentiation - lymphatic metastases - orphan receptors
