Abstract

Activation of macrophages is a hallmark of atherosclerosis. Stimulation of human monocytic leukemia THP-1 cells with phorbol 12-myristate 13-acetate (PMA) is known to induce a variety of genes whose function is relevant to activated macrophages. Flt-1, a receptor tyrosine kinase for vascular endothelial growth factor, is expressed in macrophages as well as in endothelial cells and mediates the biological response to vascular endothelial growth factor. In this study, we investigated the molecular mechanisms underlying the inducible expression of the flt-1 gene during the activation of THP-1 cells. Reverse transcription-polymerase chain reaction analysis showed that exposure of THP-1 cells to PMA increases flt-1 mRNA and protein levels. A transfected reporter gene, consisting of the human flt-1 promoter region coupled to the luciferase gene, indicated a direct effect of PMA on transcriptional activity. Transfection analysis of a series of 5'-deletion constructs and site-directed mutants localized the PMA-responsive region to a DNA stretch from -174 to -166, which represents overlapping Egr-1/Sp1 transcription factor-binding sites. Competitive gel mobility shift assays and supershift assays showed that PMA induces the binding of Egr-1 to this site. Consistent with these findings, the Egr-1 expression plasmid strongly induced flt-1 promoter activity in a sequence-specific manner. Taken together, our data demonstrate that PMA induces flt-1 gene transcription through an induction of Egr-1 in THP-1 cells, thus providing new evidence that the flt-1 gene is a direct target of Egr-1, the transcription factor primarily induced on macrophage differentiation.