Native and Oxidized Low Density Lipoprotein Induction of Tissue Factor Gene Expression in Smooth Muscle Cells Is Mediated by Both Egr-1 and Sp1*

Abstract

Tissue factor, in association with factor VIIa, initiates the coagulation cascade. We studied the influences of two pathophysiological stimuli, native (unmodified) and oxidized low density lipoprotein, on tissue factor gene expression in a cell important in vascular remodeling and vascular diseases, the smooth muscle cell. Our results demonstrated that both lipoproteins significantly induced tissue factor gene expression in rat aortic smooth muscle cells; oxidized low density lipoprotein was slightly more potent. Both lipoproteins increased tissue factor mRNA in a concentration- and time-dependent manner. Results from nuclear run-on assays and mRNA stability experiments indicated that increased tissue factor mRNA accumulation in response to the lipoproteins was principally controlled at the transcriptional level. By using lipid extracts of low density lipoprotein or methylation of the intact lipoprotein to block receptor recognition, we showed that this lipoprotein induced tissue factor mRNA via both receptor-independent and receptor-augmented pathways. Transfection studies using a series of deleted tissue factor promoters revealed that a −143- to +106-base pair region of the rat tissue factor promoter contained regulatory elements required for lipoprotein-mediated induction. Electrophoretic mobility shift assays showed that the binding activities of the transcription factor Egr-1, but not Sp1, were markedly elevated in response to these lipoproteins. Transfection of site-directed mutants of the tissue factor (TF) promoter demonstrated that not only Egr-1 but also Sp1 cis-acting elements in the TF (−143) promoter construct were necessary for optimal TF gene induction. Our data show for the first time that both low density lipoprotein and oxidized low density lipoprotein induce tissue factor gene expression in smooth muscle cells and that this tissue factor gene expression is mediated by both Egr-1 and Sp1 transcription factors.