PROX1 regulation of endothelial cell cycle progression

Abstract

The homeobox transcription factor PROX1 is necessary for proper lymphatic vessel development. PROX1 upregulates the expression of CYCLIN E1 in both Drosophila melanogaster and mammalian cells. Expression of CYCLIN E1 is controlled by two E2F sites located on either side of the transcriptional start site. We have shown that PROX1 activates a 1 kb Cyclin E1 promoter via a DNA‐binding independent mechanism that requires both E2F sites.We used reporter gene assays to assess PROX1 regulation of Cyclin E1 promoter versions in which the E2F sites were mutated. We infected Human Umbilical Vein Endothelial Cells (HUVEC) with adenovirus encoding PROX1, and used bromodeoxyuridine, proliferating cell nuclear antigen and phosphorylated histone H3 staining to quantify changes in cell cycle progression. We used western blotting to measure protein levels of CYCLIN E1.We observed a decrease in the ability of PROX1 to activate a 206 bp Cyclin E1 promoter when either E2F site was mutated. We noted an increase of CYCLIN E1 in PROX1 infected HUVECs as well as an increased number of cells in S phase. In contrast, there was not a corresponding increase of cells in M phase.We have established that both E2F sites are essential for PROX1 mediated activation of the Cyclin E1 promoter. Our studies have discovered a novel role for PROX1 as a transcriptional co‐activator.The National Cancer Institute of Canada and the Manitoba Health Research Council support this project.