Submitted on October 27, 2008
Revised on December 15, 2008
Accepted on December 29, 2008
MODULATION OF PKC
SIGNALING ALTERS THE SHEAR STRESS MEDIATED INCREASES IN ENDOTHELIAL NITRIC OXIDE SYNTHASE TRANSCRIPTION: ROLE OF STAT3
Neetu Sud1, Sanjiv Kumar1, Stephen Wedgwood2, and Stephen M Black1*
1 Medical College of Georgia
2 Northwestern University
* To whom correspondence should be addressed. E-mail: sblack{at}mcg.edu.
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 protein kinase C delta (PKC
). In this study, we explore the role of PKC in regulating shear-induced upregulation of eNOS. We found that shear caused a decrease in PKC activation. Modulation of PKC prior to shear with a dominant negative mutant of PKC (DN PKC) or bryostatin (a known PKC activator) demonstrated that PKC inhibition potentiates the shear mediated increases in eNOS expression and activity while PKC activation inhibited these events. To gain insight into the mechanism by which PKC inhibits shear induced eNOS expression, we examined activation of signal transducer and transactivator-3 (STAT3), a known target for PKC phosphorylation. We found that shear decreased the phosphorylation of STAT3. Further the transfection of cells with DN PKC reduced-, while PKC activation enhanced-, STAT3 phosphorylation in the presence of shear. Transfection of cells with a dominant negative mutant of STAT3 enhanced eNOS promoter activity and NO 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 this was no longer inhibited by bryostatin. In conclusion, shear decreases PKC 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.
