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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:e1.)
© 2002 American Heart Association, Inc.

Vascular Biology

Haplotype-Specific Effects on Endothelial NO Synthase Promoter Efficiency

Modifiable by Cigarette Smoking

Jian Wang; Donald Dudley; Xing Li Wang

From the Department of Genetics (J.W., X.L.W.), Southwest Foundation for Biomedical Research, and Department of Obstetrics and Gynecology (D.D.), University of Texas Health Science Center–San Antonio.

Correspondence to Dr. X.L. Wang, Department of Genetics, Southwest Foundation for Biomedical Research, PO Box 760549, San Antonio, TX 78245-0549. E-mail xwang{at}darwin.sfbr.org

The T–786C promoter and 27-bp repeat intron 4 polymorphisms in the endothelial NO synthase (eNOS) gene have been inconsistently associated with various eNOS-related phenotypic changes. We explored molecular mechanisms underlying the inconsistency. We constructed pGL3 luciferase reporter vectors by inserting an eNOS promoter fragment containing either T or C nucleotide at -786 bp at the 5' end of the luciferase coding region and eNOS intron 4 containing either 5x or 4x27-bp repeats at the 3' end of the luciferase gene. The transcription efficiency in the T promoter was lower than in the C promoter (15.7±1.0% vs 83.3±5.8%, P<0.01 when 5x27-bp was an enhancer and 37.6±4.7% vs 58.9±7.5%, P<0.01 when 4x27bp was an enhancer). Although cigarette smoking extracts treatment increased the transcription efficiency significantly in the T promoter (1.7-fold, P<0.01), it reduced the C promoter efficiency (by 10% to 15%). A mobility shift assay revealed positive binding of the 27-bp repeat fragment with endothelial cell nuclear protein extracts. Our study demonstrates a cis-acting role of the 27-bp repeats in eNOS promoter function and a haplotype-specific expression pattern determined by DNA variants at -786 bp and intron 4 of the eNOS gene that is also modifiable by cigarette smoking.

Key Words: gene • gene regulation • smoking • haplotypes • endothelial NO synthase

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