October 1, 1999Circulation Research82 citationsOpen Access
Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase During Cell Growth
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Key Result
Proliferating endothelial cells exhibited a 4-fold increase in eNOS mRNA levels compared to postconfluent cells, driven by mRNA stabilization via a 43-nt 3'-UTR region and a 51-kDa cytosolic protein.
Structured PICO
P
Population
Endothelial cells (proliferating vs postconfluent/nonproliferating)
I
Intervention
Transfection with chimeric cDNA plasmid containing chloramphenicol acetyl transferase (CAT) ligated to the eNOS coding region and a mutant 3'-UTR lacking a 43-nt sequence (pcDNACAT/eNOS((DeltaUTR)))
C
Comparator
Transfection with wild-type 3'-UTR (pcDNACAT/eNOS((wtUTR))) and comparison between proliferating vs postconfluent cells
O
Outcome
eNOS mRNA half-life and expression levelssurrogate
This study identifies a specific 43-nt region in the 3'-UTR of eNOS mRNA that binds a 51-kDa cytosolic protein, demonstrating a posttranscriptional mechanism for destabilizing eNOS mRNA and modulating its expression during endothelial cell growth.
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((DeltaUTR))). 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 approximately 51-kDa cytosolic protein.