Differential transcriptional regulation of the human thrombin receptor gene by the Sp family of transcription factors in human endothelial cells.
The mitogenic effects of thrombin are mediated by a G-protein-coupled receptor. Because the effects of thrombin are strongly influenced by the expression of its receptor, an understanding of its regulatory mechanisms 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 expression. 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 that 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 this 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 abilities of Sp1 and Sp3 to activate or repress HTR transcription, we conducted co-transfection experiments with wild-type or mutated HTR-promoter-luciferase constructs. Co-transfection with Sp1 significantly augmented wild-type HTR promoter activity. Sp3 alone did not affect activity, and inhibited Sp1-mediated activation. Competition for shared binding sites by Sp1 and Sp3 might differentially regulate HTR expression in vascular endothelial cells.
Wu
Y
Y
Division of Cardiology, University of Texas Medical Branch at Galveston, 301 University Boulevard, 9.138 Medical Research Building, Galveston, TX 77555-1064, USA.
Ruef
J
J
Rao
G N
GN
Patterson
C
C
Runge
M S
MS
D001011:Aorta; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008833:Microcirculation; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D018179:Receptors, Thrombin / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000096:biosynthesis / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D014158:Transcription, Genetic; D014471:Umbilical Veins
D004268:DNA-Binding Proteins; D018179:Receptors, Thrombin; D011993:Recombinant Fusion Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D008156:Luciferases