© 1999 American Heart Association, Inc.
Molecular Medicine |
Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase During Cell Growth
From the Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Ga.
Correspondence to Santhini Ramasamy, PhD, and reprint requests to Charles D. Searles, MD, Division of Cardiology, Department of Medicine, Emory University, 1639 Pierce Dr, Atlanta, GA 30322. E-mail sramasamy@issiinc.com (S.R.) or csearle{at}emory.edu (C.D.S.)
Abstract—The expression of the
endothelial NO synthase (eNOS) is dramatically
influenced by the state of cell growth. In proliferating cells, mRNA
levels are increased 4-fold compared with postconfluent,
nonproliferating cells. Nuclear run-on analysis indicated that
there is no difference in the transcriptional rate of eNOS in
proliferating versus postconfluent cells. The half-life of eNOS mRNA,
measured after actinomycin D transcriptional arrest, was 3-fold greater
in preconfluent compared with confluent endothelial
cells. Using UV–cross-linking analysis, a cytoplasmic protein
with an apparent molecular mass of 51 kDa was found to bind to
terminal 545-nt eNOS mRNA 3-fold more in confluent cells than in
proliferating cells. Further characterization of the eNOS mRNA
indicated that a 43-nt sequence at the origin of the 3'-untranslated
region (UTR) is critical in binding of this protein.
Endothelial cells were stably transfected with a
chimeric cDNA plasmid containing chloramphenicol acetyl transferase
(CAT) ligated to the eNOS coding region and either the wild-type 3'-UTR
(pcDNACAT/eNOS(wtUTR)) or a mutant 3'-UTR lacking the 43 nt
found to bind the 51-kDa protein (pcDNACAT/eNOS(
UTR)).
The CAT/eNOS mRNA half-life was dramatically stabilized in these latter
cells as compared with cells transfected with
pcDNACAT/eNOS(wtUTR)). Thus, this 43-nt region plays a
critical role in destabilizing eNOS mRNA. These studies demonstrate a
mechanism for modulation of eNOS expression during cell growth via
interactions between the proximal 3'-UTR and a novel 
51-kDa
cytosolic protein.
Key Words: mRNA stability • proliferation • RNA binding protein • 3'-untranslated region • gene regulation
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