Shear stress stimulates nitric oxide signaling in pulmonary arterial endothelial cells via a reduction in catalase activity: role of protein kinase C{delta}

Pulmonary Vascular Disease Program, Vascular Biology Center, Medical College of Georgia, Augusta, Georgia Submitted 20 August 2009 ; accepted in final form 29 October 2009 Previous studies have indicated that acute increases in shear stress can stimulate endothelial nitric oxide synthase (eNOS) activity through increased PI3 kinase/Akt signaling and phosphorylation of Ser1177. However, the mechanism by which shear stress activates this pathway has not been adequately resolved nor has the potential role of reactive oxygen species (ROS) been evaluated. Thus, the purpose of this study was to determine if shear-mediated increases in ROS play a role in stimulating Ser1177 phosphorylation and NO signaling in pulmonary arterial endothelial cells (PAEC) exposed to acute increases in shear stress. Our initial studies demonstrated that although shear stress did not increase superoxide levels in PAEC, there was an increase in H 2 O 2 levels. The increases in H 2 O 2 were associated with a decrease in catalase activity but not protein levels. In addition, we found that acute shear stress caused an increase in eNOS phosphorylation at Ser1177 phosphorylation and a decrease in phosphorylation at Thr495. We also found that the overexpression of catalase significantly attenuated the shear-mediated increases in H 2 O 2 , phospho-Ser1177 eNOS, and NO generation. Further investigation identified a decrease in PKC activity in response to shear stress, and the overexpression of PKC attenuated the shear-mediated decrease in Thr495 phosphorylation and the increase in NO generation, and this led to increased eNOS uncoupling. PKC overexpression also attenuated Ser1177 phosphorylation through a posttranslational increase in catalase activity, mediated via a serine phosphorylation event, reducing shear-mediated increases in H 2 O 2 . Together, our data indicate that shear stress decreases PKC activity, altering the phosphorylation pattern catalase, leading to decreased catalase activity and increased H 2 O 2 signaling, and this in turn leads to increases in phosphorylation of eNOS at Ser1177 and NO generation. cell signaling; phosphorylation; endothelial cell; biomechanical forces Address for reprint requests and other correspondence: S. M. Black, Vascular Biology Center: CB-3210B, Medical College of Georgia, 1459 Laney Walker Blvd., Augusta, GA 30912 (e-mail: sblack{at}mcg.edu ).