SHEAR-STRESS INDUCTION OF THE TISSUE FACTOR GENE

Using flow channel, we report that the application of a laminar shear stress induced a transient increase of tissue factor (TF) procoagulant activity in human umbilical vein endothelial cells (HUVEC), which was accompanied by a rapid and transient induction of the TF mRNA in the HUVEC. Functional analysis of the 2.2 kb TF 5' promoter indicated that a GC-rich region containing three copies each of the EGR-1 and Spl si tes was required for induction. Mutation of the Spl sites, but not the EGR-1 sites, attenuated the response of TF promoter to shear stress. Thus, Spl is a newly defined shear stress responsive element. Electrop horetic mobility shift assays showed there was no increase in binding of nuclear extracts from sheared cells to an Spl consensus site. In co ntrast, immunoblotting of these nuclear extracts with antibody against transcription factor Spl demonstrated that shear stress increased the phosphorylation of Spl. We also showed that shear stress, like the ph osphatase inhibitor okadaic acid, increased the transcriptional activi ty of Spl. These findings suggest that the shear stress induction of T F gene expression is mediated through an increased Spl transcriptional activity with a concomitant hyperphosphorylation of Sp1.