title: Up-regulation of protein kinase C-epsilon promotes the expression of cytokine-inducible nitric oxide synthase in RAW 264.7 cells.
abstract: Stimulation of the murine macrophage RAW 264.7 cell line with phorbol esters fails to promote nitric oxide synthesis as occurs in rat hepatocytes or peritoneal macrophages. Transfection of RAW 264.7 cells with plasmids harboring protein kinase C (PKC) -epsilon isotype but not with PKC-alpha, -beta1, -delta, or constitutively active -alpha and -beta1 isotypes resulted in the expression of nitric oxide synthase type II (iNOS), as reflected by the synthesis of nitric oxide measured in the culture medium of transfected cells. cotransfection of RAW 264.7 cells with the -1592 to +121-base pair promoter region of the murine iNOS gene and PKC isotypes specifically induced the transactivation of this promoter in the case of the plasmids containing the PKC-epsilon isotype. The mechanism by which PKC-epsilon induced iNOS expression involved the activation of nuclear factor binding to kappaB sites (NF-kappaB) as deduced by the suppressive effect of pyrrolidine dithiocarbamate on nitric oxide synthesis, an inhibitor of NF-kappaB activation, and by the activation of kappaB sites in cells transfected with a vector containing a kappaB motif linked to a chloramphenicol acetyltransferase reporter gene. These results suggest that PKC-epsilon can regulate a pathway that promotes iNOS expression in macrophages in response to phorbol ester activation.
authors.affiliation: Instituto de Bioquímica (Consejo Superior de Investigaciones Cientifícas), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: M J
authors.initials: MJ
authors.lastname: Díaz-Guerra
authors.forename: O G
authors.initials: OG
authors.lastname: Bodelón
authors.forename: M
authors.initials: M
authors.lastname: Velasco
authors.forename: R
authors.initials: R
authors.lastname: Whelan
authors.forename: P J
authors.initials: PJ
authors.lastname: Parker
authors.forename: L
authors.initials: L
authors.lastname: Boscá
mesh_terms: D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D007527:Isoenzymes / Q000378:metabolism*; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D051381:Rats; D014162:Transfection; D015854:Up-Regulation
chemical_list: D007527:Isoenzymes; D016328:NF-kappa B; D019001:Nitric Oxide Synthase; D015500:Chloramphenicol O-Acetyltransferase; C497256:Prkce protein, mouse; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon