Transcriptional Regulation of the lymphangiogenic factor VEGF-D: critical role of orphan receptors and promoter acetylation

Abstract

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