Induction of VEGF gene expression by retinoic acid through Sp1-binding sites in retinoblastoma Y79 cells.

PURPOSE Vascular endothelial growth factor (VEGF) is an angiogenic peptide that has been implicated in many retinopathies. Although all trans-retinoic acid (atRA) has long been known as an essential factor in the visual cycle, the role of atRA in the pathogenesis of retinal disease remains elusive. In this study, we investigated the effects of atRA on expression of the VEGF gene in retinoblastoma Y79 cells. METHODS Total RNA prepared from Y79 cells, with or without atRA, was subjected to Northern blot analyses. Reporter constructs consisting of the VEGF promoter-luciferase gene were transfected into Y79 cells. Nuclear factors binding to the VEGF promoter were analyzed by electrophoretic mobility shift assays (EMSAs). RESULTS The levels of VEGF transcripts were increased by atRA in a time- and dose-dependent manner. Progressive deletion and site-specific mutation analyses indicated that atRA increased VEGF promoter activity through a G+C-rich sequence that was shown to be an Sp1-binding site by supershift assays. EMSAs showed that Sp1 binding was increased by atRA stimulation. Although no measurable change was observed in Sp1 mRNA levels, Western blot analysis showed an increase in Sp1 protein levels in the atRA-treated cells. These data suggest that atRA increases Sp1 protein levels by posttranscriptional mechanisms, and elevated levels of Sp1 protein induce the expression of VEGF at the transcriptional level. CONCLUSIONS atRA induced transcription of the VEGF gene through Sp1-binding sites in Y79 cells. Pharmacologic intervention that inhibits the signals elicited by atRA may be effective in treating VEGF-mediated retinopathies.