Responsive site on the thrombospondin-1 promotor to down-regulation by phorbol 12-myristate 13-acetate in porcine aortic endothelial cells

Abstract

Thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, inhibits neovascularization and is implicated in the regression of tumor growth and metastasis. We found that the synthesis of TSP-1 in porcine aortic endothelial (PAE) cells was decreased in a dose-dependent manner by phorbol 12-myristate 13-acetate (PMA) treatment in porcine aortic endothelial (PAE) cells. In this study, a responsive site on the TSP-1 promotor affected by PMA treatment in PAE was characterized. The level of TSP-1 mRNA was also decreased by PMA after 1 h and persisted that way for at least 24 h. PMA treatment and c-Jun overexpression suppressed the transcription of TSP-1 promotor-luciferase reporter gene. A deletion between -767 and -657 on the TSP-1 promotor neutralized the PMA-induced down-regulation. In addition, oligo a (-767 ∼-723) was responsive to PMA-induced repression, while oligo b (-734 ∼-689) and c (-700 ∼-656) was not. Electrophoretic mobility shift assays showed that this PMA responsive element specifically bound a nuclear protein and that the binding activity was diminished by PMA treatment in PAE cells but not in Hep 3B cells. In supershift assay, potential regulatory elements in this region, SP1 and GATA-1, were not responsive to the inhibition of TSP-1 expression by PMA. Our results suggest that the repression of TSP-1 synthesis by PMA is mediated by blocking a particular unknown nuclear protein binding to the responsive site (-767 ∼-735), which is regulated by c-Jun.