DIFFERENTIAL TRANSCRIPTIONAL REGULATION OF THE HUMAN THROMBIN RECEPTOR GENE BY THE SP FAMILY OF TRANSCRIPTION FACTORS IN HUMAN ENDOTHELIAL-CELL

The mitogenic effects of thrombin are mediated by a G-protein-coupled receptor. Because the effects of thrombin are strongly influenced by t he expression of its receptor, an understanding of its regulatory mech anisms is essential. To identify mechanisms of human thrombin receptor (HTR) gene regulation, a series of HTR-promoter-luciferase constructs were made and transfected into human microvascular endothelial cells for analysis. Deletion from bp -303 to -164 abolished reporter gene ex pression. Dimethyl sulphate treatment in vivo and DNase I footprinting in vitro demonstrated that a cluster of three GC box consensus sites was occupied, and electrophoretic mobility-shift assays established th at Sp1 and Sp3 both bind to this 3' GC box cluster. We mutated each of the three GC boxes individually and all three collectively within thi s 3' cluster. Basal promoter activity was decreased to 46%, 78% and 29 %, of control for each of the GC boxes mutated individually, and to 6% when the three were mutated collectively. To test the individual abil ities of Sp1 and Sp3 to activate or repress HTR transcription, we cond ucted cotransfection experiments with wild-type or mutated HTR-promote r-luciferase constructs. Co-transfection with Spl significantly augmen ted wild-type HTR promoter activity. Sp3 alone did not affect activity , and inhibited Sp1-mediated activation. Competition for shared bindin g sites by Sp1 and Sp3 might differentially regulate HTR expression in vascular endothelial cells.