title: Inhibition of classical PKC isoenzymes downregulates STAT1 activation and iNOS expression in LPS-treated murine J774 macrophages.
abstract: Proinflammatory cytokines and bacterial products trigger inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in inflammatory and tissue cells. In inflammation, NO acts as an important mediator having both proinflammatory and destructive effects. Protein kinase C (PKC) is a family of serine-threonine protein kinase isoenzymes involved in signal transduction pathways related to inflammatory responses. The aim of the present study was to investigate the role of classical PKC (cPKC) isoenzymes in the regulation of iNOS expression and NO production in murine J774 macrophages and the mechanisms involved. RO318220 (inhibits PKCbeta, PKCgamma and PKCvarepsilon), GO6976 (inhibits cPKC isoenzymes PKCalpha and PKCbeta) and LY333531 (inhibits PKCbeta) reduced lipopolysaccharide (LPS)-induced NO production and iNOS expression in a dose-dependent manner as did 6 h pretreatment with 1 microM phorbol 12-myristate 13-acetate (PMA) (which was shown to downregulate PKC expression). PKC inhibitors also reduced LPS-induced iNOS mRNA levels, but they did not affect the half-life of iNOS mRNA. PKC inhibitors did not alter LPS-induced activation of NF-kappaB as measured by electrophoretic mobility shift assay. All PKC inhibitors used and pretreatment with 1 microM PMA inhibited signal transducer and activator of transcription 1 (STAT1) activation as measured by the translocation of STAT1alpha from the cytosol to the nucleus by Western blot. In addition, inhibition of STAT1 activation by AG-490, an inhibitor of JAK-2, also reduced NO production. These results suggest that cPKC isoenzymes, especially PKCbeta, mediate the upregulation of iNOS expression and NO production in activated macrophages in an NF-kappaB-independent manner, possibly through the activation of transcription factor STAT1.
authors.affiliation: The Immunopharmacology Research Group, Medical School, University of Tampere and Research Unit, Tampere University Hospital, Tampere, Finland.
authors.forename: Tiina
authors.initials: T
authors.lastname: Salonen
authors.forename: Outi
authors.initials: O
authors.lastname: Sareila
authors.forename: Ulla
authors.initials: U
authors.lastname: Jalonen
authors.forename: Hannu
authors.initials: H
authors.lastname: Kankaanranta
authors.forename: Raimo
authors.initials: R
authors.lastname: Tuominen
authors.forename: Eeva
authors.initials: E
authors.lastname: Moilanen
mesh_terms: D000818:Animals; D001711:Biotransformation / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D007527:Isoenzymes / Q000378:metabolism; D053614:Janus Kinase 2; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D009573:Nitrites / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D050794:STAT1 Transcription Factor / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009573:Nitrites; D010703:Phorbol Esters; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D052247:Nitric Oxide Synthase Type II; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D011493:Protein Kinase C