Abstract 860: Statins Modulate eNOS mRNA Processing
HMG-CoA reductase inhibitors (statins) are potent cholesterol-lowering drugs, but they also have pleiotropic properties that enable them to exert beneficial biological effects on endothelial function aside from their lipid-lowering properties. One of these effects is increased endothelial nitric oxide synthase (eNOS) expression, which occurs predominantly through posttranscrip-tional mechanisms involving enhanced mRNA stability. Recently, we described a novel mechanism for posttranscriptional regulation of eNOS that involves changes in 3′ poly(A) tail length of eNOS mRNA. The 3′ poly(A) tail is important in regulating mRNA stability and translational efficiency. The purpose of this study was to characterize changes in eNOS mRNA 3′ polyadenylation in response to statins. Using ribonuclease protection assays with a riboprobe targeted to the 3′ end of eNOS mRNA, we found that treatment with rosuvastatin caused a time and dose-dependent increase in 3′ polyadenylation of eNOS mRNA in bovine aortic endothelial cells. Cells treated for 24 hours with rosuvastatin (10 μM) had a 4.92-fold (p < 0.05) increase in the ratio of long (75 160 nt) to short (<25 nt) eNOS transcripts compared to untreated cells. Simvastatin induced similar effects on eNOS 3′ polyadenylation at higher concentrations (2.95-fold increase of long/short eNOS transcripts vs control, p<0.05). In DRB chase studies, polyadenylated eNOS mRNA from statin-treated cells was dramatically more stable than that from untreated cells. Statin-induced 3′ polyadenylation was inhibited by the isoprenoids mevalonate and geranylgeranyl pyrophosphate by 64% and 75%, respectively (p<0.05 for either treatment), but farnesyl pyrophosphate co-treatment had no significant effect. Further, we found that statin-induced changes in eNOS mRNA 3′ polyadenylation occurred in the nucleus, before export into the cytosol. Finally, we found that statin increased phosphorylation of RNA polymerase II, suggesting that statins exert their effects on eNOS 3′ processing by modulating the activity of RNAP II, the enzyme chiefly responsible for mRNA processing. Together, our data support the concept that statins modulate eNOS expression via a novel mechanism that involves changes in mRNA 3′ polyadenylation.