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Laminar Shear Stress Modulates Phosphorylation and Localization of RNA Polymerase II on the Endothelial Nitric Oxide Synthase Gene

Objective—In endothelial cells exposed to unidirectional laminar shear stress, endothelial nitric oxide synthase transcription (eNOS), mRNA stability, and protein levels are enhanced. We have previously demonstrated that these changes are associated with increased 3′ polyadenylation of eNOS mRNA. Here, we investigated the effect of laminar shear stress on the phosphorylation and localization of RNA polymerase (Pol) II, the enzyme primarily responsible for coordinating transcription and posttranscriptional processing. Methods and Results—Using Western and chromatin immunoprecipitation analyses, Pol II phosphorylation and localization on the eNOS gene were assessed in bovine aortic endothelial cells exposed to laminar shear stress. Total Pol II (phosphorylated and unphosphorylated) levels were increased 65% in response to laminar shear stress. This was associated with an increase in Pol II phosphoserine 2, but no change in levels of the unphosphorylated or phosphoserine 5 isoforms. Quantitative chromatin immunoprecipitation analysis showed that laminar shear stress enhanced binding of Pol II phosphoserine 2 to the 3′ end of the eNOS gene, particularly exon 26, which encodes the 3′UTR. Treatment of cells with DRB attenuated laminar shear stress-induced Pol II phosphorylation, eNOS 3′ polyadenylation, and eNOS expression. Conclusion—These data suggest that laminar shear stress enhances eNOS mRNA 3′ polyadenylation by modulating phosphorylation and localization of Pol II.