Thrombin induces Egr-1 promoter activity in primary endothelial cells.

Primary human endothelial cells were transiently transfected with 0.5 μg of Egr-1 promoter reporter gene constructs, serum-starved in EBM-2 medium containing 0.5% FBS for 18 hours, treated in the absence or presence of 1.5 U/mL thrombin for 6 hours, and then harvested for luciferase activity. The results show the means and standard deviations of luciferase light units (relative to control untreated cells) obtained in triplicate from 4 independent experiments. Luciferase light units were corrected for transfection efficiency as described in “Materials and methods.” (A) HPAECs, HCAECs, or HUVECs were transiently transfected with the full-length wild-type 1200-bp Egr-1-Luc plasmid. (B) HUVECs were transiently transfected with a series of 5′ or internal deletion mutants of the Egr-1promoter coupled to a luciferase reporter gene (pXP2). Sequences are numbered relative to the start site of transcription. The SREs are shown in bars. (C) HUVECs were transiently transfected with SRE heterologous promoter constructs, in whichEgr-1 promoter fragments encompassing the 5′ SRE cluster, 3′ SRE cluster, or both clusters were fused upstream of a minimalvon Willebrand factor (VWF) core promoter as previously described.28 For all panels, *P < .01, compared with control untreated cells.