期刊
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
卷 25, 期 12, 页码 2509-2514出版社
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/01.ATV.0000189306.99112.4c
关键词
endothelial nitric oxide synthase; intron 4; nuclear beta-actin
资金
- NHLBI NIH HHS [R01 HL066053-04, R01 HL066053, R01-HL066053] Funding Source: Medline
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 beta-actin was one of the major 27nt binding proteins. Using the pGL3 reporter vectors containing the 5 x 27nt repeats as an enhancer in an in vitro transcription assay, we found that exogenous beta-actin significantly increased reporter gene transcription efficiency. The beta-actin's upregulating effect was compromised when exogenous 27nt RNA duplex was added. Furthermore, the eNOS expression was reduced when beta-actin gene was silenced by specific siRNA, and actin overexpression upregulated eNOS expression > 3-fold. Conclusion-Our data demonstrate that beta-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.
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