Role of Sp1 in transcriptional activation of human nitric oxide synthase type III gene.

Endothelial nitric oxide synthase (eNOS or NOS-III) is constitutively expressed. To elucidate the mechanism by which the basal expression of NOS-III gene is activated, we constructed in a luciferase vector, pXP1, serial 5'-deletion mutants of a 1.3-kb 5'-flanking fragment and transiently expressed them in cultured human endothelial cells. The promotor activity was detected in the -198/+22 region which contains several putative Sp1 binding sites. DNase I footprinting assays coupled with gel shift assays revealed the GC box(-104/-90) to be the Sp1 binding site. Site-directed mutation of 4 crucial bases in this site reduced the promotor activity by > 90%. These findings provide strong evidence that binding of Sp1 or closely related protein to this site is required for the activation of basal NOS-III transcription.