Shear stress induces expression of vascular endothelial growth factor receptor Flk-1/KDR through the CT-rich Sp1 binding site.
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/cm2) elevated Flk-1/KDR mRNA levels by approximately 3-fold for 8 hours, and the expression was upregulated within the range of 5 to 40 dyne/cm2. 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-kappaB, 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.
Abumiya
Takeo
T
National Cardiovascular Center Research Institute, Osaka, Japan. abumiya@mbh.nifty.com
Sasaguri
Toshiyuki
T
Taba
Yoji
Y
Miwa
Yoshikazu
Y
Miyagi
Megumi
M
D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D003596:Cytosine / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D016228:Endothelial Growth Factors / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D008222:Lymphokines / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis*; D017978:Receptors, Growth Factor / Q000096:biosynthesis*; D011975:Receptors, Mitogen / Q000096:biosynthesis*; D040262:Receptors, Vascular Endothelial Growth Factor; D012212:Rheology; D013314:Stress, Mechanical; D013941:Thymine / Q000502:physiology*; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D008222:Lymphokines; D017978:Receptors, Growth Factor; D011975:Receptors, Mitogen; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D003596:Cytosine; D011494:Protein Kinases; C065999:Sp1 kinase; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D013941:Thymine