title: Specific PKC isoforms regulate LPS-stimulated iNOS induction in murine microglial cells.
abstract: BACKGROUND
  Excessive production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) in reactive microglia is a major contributor to initiation/exacerbation of inflammatory and degenerative neurological diseases. Previous studies have indicated that activation of protein kinase C (PKC) can lead to iNOS induction. Because of the existence of various PKC isoforms and the ambiguous specificity of PKC inhibitors, it is unclear whether all PKC isoforms or a specific subset are involved in the expression of iNOS by reactive microglia. In this study, we employed molecular approaches to characterize the role of each specific PKC isoform in the regulation of iNOS expression in murine microglia.
  
  
  METHODS
  Induction of iNOS in response to bacterial endotoxin lipopolysaccharide (LPS) was measured in BV-2 murine microglia treated with class-specific PKC inhibitors, or transfected with siRNA to silence specific PKC isoforms. iNOS expression and MAPK phosphorylation were evaluated by western blot. The role of NF-κB in activated microglia was examined by determining NF-κB transcriptional response element- (TRE-) driven, promoter-mediated luciferase activity.
  
  
  RESULTS
  Murine microglia expressed high levels of nPKCs, and expressed relatively low levels of cPKCs and aPKCs. All PKC inhibitors attenuated induction of iNOS in LPS-activated microglia. Knockdown of PKC δ and PKC β attenuated ERK1/2 and p38 phosphorylation, respectively, and blocked NF-κB activation that leads to the expression of iNOS in reactive microglia.
  
  
  CONCLUSIONS
  Our results identify PKC δ and β as the major PKC isoforms regulating iNOS expression in reactive microglia. The signaling pathways mediated by PKC involve phosphorylation of distinct MAPKs and activation of NF-κB. These results may help in the design of novel and selective PKC inhibitors for the treatment of many inflammatory and neurological diseases in which production of NO plays a pathogenic role.
authors.affiliation: Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Wen
authors.forename: Rachel
authors.initials: R
authors.lastname: Ribeiro
authors.forename: Yumin
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D004790:Enzyme Induction; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000502:physiology; D051379:Mice; D017628:Microglia / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009573:Nitrites; D034741:RNA, Small Interfering; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases