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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:2509.)
© 2005 American Heart Association, Inc.

Vascular Biology

Effect of Nuclear Actin on Endothelial Nitric Oxide Synthase Expression

Hesheng Ou; Ying H. Shen; Budi Utama; Jian Wang; Xinwen Wang; Joseph Coselli; Xing Li Wang

From the Section of Adult Cardiothoracic Service, Texas Heart Institute at St Luke’s Episcopal Hospital, and the Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Tex.

Correspondence to Xing Li Wang, NAB 2010, One Baylor Plaza, Baylor College of Medicine, Houston, TX 77030. E-mail xlwang{at}bcm.tmc.edu

Background— Previously, we showed that the 27nt repeat polymorphism in endothelial nitric oxide synthase (eNOS) intron 4 was associated with altered eNOS mRNA and protein levels, nitric oxide (NO) production and vascular disease risk; the 27-nt repeats had a cis-acting role in eNOS promoter function. In the present study, we investigated nuclear protein that binds the 27nt repeat and mediates eNOS expression.

Methods and Results— Using 5'-biotin-labeled 27nt DNA duplex and streptavidin–agarose beads pull-down assay and mass spectrometry, we identified that nuclear ß-actin was one of the major 27nt binding proteins. Using the pGL3 reporter vectors containing the 5x27nt repeats as an enhancer in an in vitro transcription assay, we found that exogenous ß-actin significantly increased reporter gene transcription efficiency. The ß-actin’s upregulating effect was compromised when exogenous 27nt RNA duplex was added. Furthermore, the eNOS expression was reduced when ß-actin gene was silenced by specific siRNA, and actin overexpression upregulated eNOS expression >3-fold.

Conclusion— Our data demonstrate that ß-actin as a transcription factor stimulates eNOS expression; and the transcriptional effect appears to be 27nt-dependent. Our findings represent a novel molecular mechanism regulating eNOS expression, which could potentially lead to discoveries of eNOS specific pharmaceutical agents, eg, active peptides, with clinical applications.

The 27nt repeat element in eNOS intron 4 functions as a cis-acting enhancer regulating eNOS expression. We found that nuclear actin binds on the 27nt DNA fragment and upregulates eNOS expression. Whereas nuclear actin may upregulate several other genes, its effect on eNOS depends on the presence of the 27nt enhancer

Key Words: endothelial nitric oxide synthase • intron 4 • nuclear ß-actin

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