An important vascular adaptation to laminar shear stress is increased expression of the endothelial cell nitric oxide synthase (eNOS). This not only occurs in cultured endothelial cells, but it also occurs in vessels exposed to high levels of flow, such as during exercise training. Research from our laboratory has shown that eNOS upregulation in response to shear stress occurs via two divergent pathways, both of which are dependent on the tyrosine kinase cSrc. One pathway leads to a brief one hour increase in transcription that is dependent on a classical ERK1/2 pathway. Following this, continued shear stress leads to a prolonged stabilization of the eNOS mRNA that is independent of ERK1/2 or its upstream signals. The mechanisms regulating eNOS mRNA stability remain poorly defined. In preliminary studies, we have found that laminar shear stress dramatically increases the 3' polyadenylation of eNOS transcripts; eNOS 3' poly(A) tails of 75 nt to 160 nt are expressed in response to shear stress compared to poly(A) tails of <25 nt under basal conditions. ENOS transcripts with longer poly(A) tails are more stable and are translationally more active than those with short poly(A) tails. This modulation of RNA 3' processing seems to represent a completely novel mechanism for regulation of gene expression and translation in response to mechanical stimuli. In the proposed studies, we plan to gain additional insight into this phenomenon.
In aim one, we will identify the hexanucleotide element in the eNOS polyadenylation signal and determine its role in shear-induced polyadenylation. We will examine the polyadenylation and cleavage efficiency of eNOS constructs containing wild-type sequence element and compare it to eNOS constructs with canonical sequence element. This will be studied using in vitro assays and in eNOS-/- cells exposed to shear.
In aim 2, we will examine conserved sequences upstream to the eNOS poly(A) signal for their responsiveness to shear by making sequential deletions of this region and examining polyadenylation efficiency in vitro and in cells. We have preliminary data suggesting that HMG CoA reductase inhibitors also increase eNOS 3' poly(A) tail length.
In aim 3, we will examine mechanisms responsible for this phenomenon. In this aim, we will determine the roles of the small G protein Rho, its target Rho kinase, and cytoskeleton organization in eNOS mRNA 3' poly(A) tail processing. Finally, in aim 4, we will determine if shear stress associated with exercise training modulates eNOS 3' polyadenylation in vivo. In these experiments, we will exercise train wild-type mice and mice heterozygotic for cSrc (in whom eNOS expression is not increased by exercise). Overall, these studies promise to provide very novel information regarding how mechanical forces and other stimuli modulate gene expression via regulation of mRNA 3' poly(A) tail length.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL077274-01A1
Application #
6923363
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Goldman, Stephen
Project Start
2005-04-01
Project End
2009-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
1
Fiscal Year
2005
Total Cost
$372,377
Indirect Cost
Name
Emory University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Moore, Jeffrey P; Weber, Martina; Searles, Charles D (2010) Laminar shear stress modulates phosphorylation and localization of RNA polymerase II on the endothelial nitric oxide synthase gene. Arterioscler Thromb Vasc Biol 30:561-7
Weber, Martina; Baker, Meredith B; Moore, Jeffrey P et al. (2010) MiR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity. Biochem Biophys Res Commun 393:643-8
Jones, Rachel; Baker, Meredith B; Weber, Martina et al. (2009) Molecular beacons can assess changes in expression and 3'-polyadenylation of human eNOS mRNA. Am J Physiol Cell Physiol 296:C498-504
Kosmidou, Ioanna; Moore, Jeffrey P; Weber, Martina et al. (2007) Statin treatment and 3'polyadenylation of eNOS mRNA. Arterioscler Thromb Vasc Biol 27:2642-9