Modulation of PKCδ signaling alters the shear stress-mediated increases in endothelial nitric oxide synthase transcription: role of STAT3

Sud N, Kumar S, Wedgwood S, Black SM. Modulation of PKC delta signaling alters the shear stress-mediated increases in endothelial nitric oxide synthase transcription: role of STAT3. Am J Physiol Lung Cell Mol Physiol 296: L519-L526, 2009. First published December 31, 2008; doi:10.1152/ajplung.90534.2008.-We have previously shown that the regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells isolated from fetal lamb under static conditions is positively regulated by PKC delta. In this study, we explore the role of PKC delta in regulating shear-induced upregulation of eNOS. We found that shear caused a decrease in PKC delta activation. Modulation of PKC delta before shear with a dominant negative mutant of PKC delta (DN PKC delta) or bryostatin (a known PKC delta activator) demonstrated that PKC delta inhibition potentiates the shear-mediated increases in eNOS expression and activity, while PKC delta activation inhibited these events. To gain insight into the mechanism by which PKC delta inhibits shear-induced eNOS expression, we examined activation of STAT3, a known target for PKC delta phosphorylation. We found that shear decreased the phosphorylation of STAT3. Further the transfection of cells with DN PKC delta reduced, while PKC delta activation enhanced, STAT3 phosphorylation in the presence of shear. Transfection of cells with a dominant negative mutant of STAT3 enhanced eNOS promoter activity and nitric oxide production in response to shear. Finally, we found that mutating the STAT3 binding site sequence within the eNOS promoter increased promoter activity in response to shear and that this was no longer inhibited by bryostatin. In conclusion, shear decreases PKC delta activity and, subsequently, reduces STAT3 binding to the eNOS promoter. This signaling pathway plays a previously unidentified role in the regulation of eNOS expression by shear stress.