Shear stress regulates endothelial nitric-oxide synthase promoter activity through nuclear factor kappaB binding.
We have previously demonstrated that shear stress increases transcription of the endothelial nitric-oxide synthase (eNOS) by a pathway involving activation of the tyrosine kinase c-Src and extracellular signal-related kinase 1/2 (ERK1/2). In the present study sought to determine the events downstream of this pathway. Shear stress activated a human eNOS promoter chloramphenicol acetyl-CoA transferase chimeric construct in a time-dependent fashion, and this could be prevented by inhibition of the c-Src and MEK1/2. Studies using electromobility shift assays, promoter deletions, and promoter mutations revealed that shear activation of the eNOS promoter was due to binding of nuclear factor kappaB subunits p50 and p65 to a GAGACC sequence -990 to -984 base pairs upstream of the eNOS transcription start site. Shear induced nuclear translocation of p50 and p65, and activation of the eNOS promoter by shear could be prevented by co-transfection with a dominant negative I kappa Balpha. Exposure of endothelial cells to shear resulted in Ikappa kinase phosphorylation, and this was blocked by the MEK1/2 inhibitor PD98059 and the cSrc inhibitor PP1, suggesting these signaling molecules are upstream of NFkappaB activation. These experiments indicate that shear stress increases eNOS transcription by NFkappaB activation and p50/p65 binding to a GAGACC sequence present of the human eNOS promoter. While NFkappaB activation is generally viewed as a proinflammatory stimulus, the current data indicate that its transient activation by shear may increase expression of eNOS, which via production of nitric oxide could convey anti-inflammatory and anti-atherosclerotic properties.
Division of Cardiology, Molecular and Systems Pharmacology Program, Emory University, Atlanta, Georgia 30322, USA.
Michael E
ME
Davis
Isabella M
IM
Grumbach
Tohru
T
Fukai
Alexis
A
Cutchins
David G
DG
Harrison
D000818:Animals; D001011:Aorta; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004730:Endothelium, Vascular; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D016296:Mutagenesis; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013314:Stress, Mechanical; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D016328:NF-kappa B; D021122:Protein Subunits; D011994:Recombinant Proteins; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III