Shear Stress Induces Expression of Vascular Endothelial Growth Factor Receptor Flk-1/KDR Through the CT-Rich Sp1 Binding Site

Abstract

Fluid shear stress is 1 of the major factors that control gene expression in vascular endothelial cells. We investigated the role of shear stress in the regulation of the expression of fetal liver kinase-1/kinase domain region (Flk-1/KDR), a vascular endothelial growth factor receptor, by using human umbilical vein endothelial cells. Laminar shear stress (15 dyne/cm 2 ) elevated Flk-1/KDR mRNA levels by ≈3-fold for 8 hours, and the expression was upregulated within the range of 5 to 40 dyne/cm 2 . Deletion analysis of the 5′-flanking region of the Flk-1/KDR gene promoter by use of a luciferase reporter vector revealed that a shear stress–responsive element resided in the sequence between −94 and −31 bp, which contained putative nuclear factor-κB, activator protein-2, and GC-rich Sp1 and CT-rich Sp1 binding sites. Electrophoretic mobility shift assay demonstrated that nuclear extract was bound to the GC-rich Sp1 sites and the CT-rich Sp1 site with a similar pattern. However, shear stress enhanced the DNA-protein interactions only on the CT-rich Sp1 site but not on the GC-rich Sp1 sites. A 3-bp mutation in the CT-rich Sp1 site eliminated the response to shear stress in electrophoretic mobility shift assay and luciferase reporter assay. These results suggest that shear stress induces Flk-1/KDR expression through the CT-rich Sp1 binding site.