Abstract 485: Activation of p300 Histone Acetyltransferase Activity is an Early Endothelial Response to Laminar Shear Stress and is Essential for Stimulation of Transcription of the Endothelial Nitric Oxide Synthase mRNA
Shear stress, the tangential force of flow over the surface of the endothelium, modulates expression of several genes including the endothelial cell nitric oxide synthase (eNOS). We have reported that stimulation of eNOS mRNA transcription by shear is dependent on a transient nuclear translocation of Nuclear Factor kappa B (NF-κB) subunits p50 and p65, and binding of these to a shear stress response element (SSRE) in the human eNOS promoter. The purpose of this study was to examine signaling events upstream of p50 and p65 DNA binding. Human umbilical vein endothelial cells were exposed to static conditions or unidirectional laminar shear (15 dynes/cm2). Using Western blots, we found modest amounts of both p50 and p65 in the nuclei of these cells at rest, and that 30 minutes of laminar shear increased nuclear p50 by 2.1± 0.1 and p65 by 3.0 ± 0.8 fold (p< 0.02 for each, n=4). Using the Chromatin Immunoprecipitation (CHIP) assay, we showed that virtually no p50 or p65 was bound to the eNOS promoter in the absence of shear, but that shear caused a robust binding of both of these subunits to the SSRE region of the eNOS promoter within 30 minutes. Shear also stimulated p65 binding to the transcriptional co-activator p300 within 10 minutes as assessed by co-immunoprecipitation. P300 has histone acetyltransferase (HAT) activity, and 20 minutes of shear increased this by 2.5 ± 0.4 fold (p < 0.001, n=5). Moreover, p65 was acetylated in response to shear within 30 minutes. These findings suggest that activation of p300 might lead to p65 acetylation, increasing its binding to the eNOS SSRE and stimulation of eNOS mRNA transcription. Indeed the p300/HAT activity inhibitor Curcumin and p300 small interfering siRNA completely abolished the increase in eNOS mRNA transcription in response to 5 hours of shear stress (n=4). Finally, using CHIP assay, we showed that shear rapidly stimulates acetylation of histones 3 and 4 at the site of the SSRE and extending 3′ toward the eNOS coding region (n=3). This was associated with opening of chromatin at the SSRE. In conclusion, these studies have revealed a previously unknown role of p300/HAT in the very early response to shear, by increasing p65 DNA binding and chromatin remodeling, both of which are essential for stimulating eNOS transcription.