title: Regulation of NADPH oxidase 5 by protein kinase C isoforms.
abstract: NADPH oxidase5 (Nox5) is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular complications of diabetes and other diseases with increased ROS production.
authors: Department of Forensic Medicine, Nanjing Medical University, Nanjing, Jiangsu, China ; Vascular Biology Center, Georgia Regents University, Augusta, Georgia, United States of America.
authors: Feng
authors: F
authors: Chen
authors: Department of Forensic Medicine, Nanjing Medical University, Nanjing, Jiangsu, China ; Vascular Biology Center, Georgia Regents University, Augusta, Georgia, United States of America.
authors: Yanfang
authors: Y
authors: Yu
authors: Vascular Biology Center, Georgia Regents University, Augusta, Georgia, United States of America.
authors: Steven
authors: S
authors: Haigh
authors: Department of Pharmacology and Toxicology, Georgia Regents University, Augusta, Georgia, United States of America.
authors: John
authors: J
authors: Johnson
authors: Vascular Biology Center, Georgia Regents University, Augusta, Georgia, United States of America ; Department of Pharmacology and Toxicology, Georgia Regents University, Augusta, Georgia, United States of America.
authors: Rudolf
authors: R
authors: Lucas
authors: Vascular Biology Center, Georgia Regents University, Augusta, Georgia, United States of America ; Department of Pharmacology and Toxicology, Georgia Regents University, Augusta, Georgia, United States of America.
authors: David W
authors: DW
authors: Stepp
authors: Vascular Biology Center, Georgia Regents University, Augusta, Georgia, United States of America ; Department of Pharmacology and Toxicology, Georgia Regents University, Augusta, Georgia, United States of America.
authors: David J R
authors: DJ
authors: Fulton
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D057809:HEK293 Cells; D006801:Humans; D006943:Hyperglycemia / Q000201:enzymology / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D013481:Superoxides; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C431571:NOX5 protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha