title: Shear stress stimulates nitric oxide signaling in pulmonary arterial endothelial cells via a reduction in catalase activity: role of protein kinase C delta.
abstract: 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 H2O2 levels. The increases in H2O2 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 H2O2, phospho-Ser1177 eNOS, and NO generation. Further investigation identified a decrease in PKCdelta activity in response to shear stress, and the overexpression of PKCdelta attenuated the shear-mediated decrease in Thr495 phosphorylation and the increase in NO generation, and this led to increased eNOS uncoupling. PKCdelta overexpression also attenuated Ser1177 phosphorylation through a posttranslational increase in catalase activity, mediated via a serine phosphorylation event, reducing shear-mediated increases in H2O2. Together, our data indicate that shear stress decreases PKCdelta activity, altering the phosphorylation pattern catalase, leading to decreased catalase activity and increased H2O2 signaling, and this in turn leads to increases in phosphorylation of eNOS at Ser1177 and NO generation.
authors.affiliation: Vascular Biology Center: CB-3210B, Medical College of Georgia, 1459 Laney Walker Blvd., Augusta, GA 30912, USA.
authors.forename: Sanjiv
authors.initials: S
authors.lastname: Kumar
authors.forename: Neetu
authors.initials: N
authors.lastname: Sud
authors.forename: Fabio V
authors.initials: FV
authors.lastname: Fonseca
authors.forename: Yali
authors.initials: Y
authors.lastname: Hou
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
mesh_terms: D000818:Animals; D002374:Catalase / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D005799:Genes, Dominant; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D050505:Mutant Proteins / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010768:Phosphoserine / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology*; D012756:Sheep; D015398:Signal Transduction / Q000187:drug effects; D013314:Stress, Mechanical; D013481:Superoxides / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D047428:Protein Kinase Inhibitors; D013481:Superoxides; D010768:Phosphoserine; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D002374:Catalase; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D051745:Protein Kinase C-delta