Abstract 255: Laminar Shear Stress Enhances the Activity and Localization of RNA Polymerase II on the eNOS Gene
We have previously found that laminar shear enhances eNOS mRNA stability and translation by altering endothelial NO synthase (eNOS) mRNA 3′ polyadenylation. Transcription is tightly coupled to pre-mRNA processing, and is coordinated by RNA polymerase (RNAP) II. We assessed whether laminar shear stress alters the activity and localization of RNAP II on the eNOS gene. We found that endothelial cells exposed to laminar shear stress had a 2-fold (n=3, p < 0.05) increase in total RNAP II protein levels compared to control; this effect was dose-dependent (0–15 dynes/cm2). Since three different RNAP II phosphoisoforms are associated with different stages of mRNA synthesis, we examined whether shear stress affected protein expression of these particular phosphoisoforms. We found that cells exposed to laminar shear had a 3-fold increase (p<0.05) in expression of RNAP II phosphorylated at serine 2, which is associated with transcription elongation and 3′ polyadenylation. In contrast, shear stress did not alter expression of the other phosphoisoforms. We performed chromatin immunoprecipitation (ChIP) analysis to examine shear-induced changes in RNAP II localization on the eNOS gene. Using antibody against total RNAP II, eNOS sequences along the entire gene were amplified. Using the phosphoserine 2 specific RNAP II antibody, we found active RNAP II predominantly bound to the 3′UTR (exon 26) and downstream sequence of eNOS in sheared cells. These findings were confirmed by quantitative ChIP (3 fold increase, n = 8, p = 0.0378). This suggests that shear-induced changes in RNAP II phosphorylation enhance its ability to polyadenylate eNOS mRNA. Serine 2 phosphorylation is dependent on cyclin-dependent kinase 9 (CDK 9), and shear-induced recruitment of CDK 9 to the eNOS gene was examined. We found that endothelial cells exposed to laminar shear had enhanced localization of CDK 9 to the eNOS promoter and exons 8 and 22, and diminished localizationto exon 26 and in the downstream sequence. This indicates that shear recruits CDK 9 while RNAP II is bound to the promoter and early exons to activate RNAP by phosphorylating serine 2. In conclusion, laminar shear stress enhances eNOS mRNA processing and increases gene expression through recruitment of CDK 9 and activation of RNAP II.