Properties of protein kinase C isoforms (beta II, epsilon, and zeta) in a macrophage cell line (J774) and their roles in LPS-induced nitric oxide production.
Northern analysis of poly(A)+ RNA extracted from J774 cells (a mouse macrophage cell line) showed that this cell line constitutively expresses mRNAs specific for protein kinase C (PKC)-beta I, -beta II, -epsilon and -zeta, but not those for PKC-alpha, -gamma or -delta. Western analysis of the total cell lysate showed that J774 cells express PKC-beta II, -epsilon and -zeta isoenzymes, but failed to show the expression of PKC-beta I. The exposure of J774 cells to > 10 nM PMA led to a loss of immunoreactive PKC-beta II in 4 h. The down-regulation of immunoreactive PKC-epsilon required more than 8 h of the exposure to > 100 nM PMA. Immunoreactive PKC-zeta was most resistant to PMA treatment and was not significantly reduced after the exposure to 300 to 600 nM PMA for 24 h. PMA-mediated, persistent down-regulation of PKC-beta II is probably a result of the inhibition of PKC-beta II biosynthesis at the posttranscriptional level, because PMA-exposed cells were found to gradually increase the expression of PKC-beta II specific mRNA. PMA-pretreated cells responded to a low dose (10 ng/ml), but not to a high dose (1 microgram/ml), of LPS by significantly lower expression of mRNA specific for the inducible nitric oxide synthase (i-NOS) gene and production of nitric oxide (NO) than the control cells did. Thus, PKC could be a part of the signal transduction apparatus involved in LPS-induced inducible nitric oxide synthase gene activation.
Fujihara
M
M
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66160.
Connolly
N
N
Ito
N
N
Suzuki
T
T
D000594:Amino Acid Oxidoreductases / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005260:Female; D007527:Isoenzymes / Q000032:analysis*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase; D011493:Protein Kinase C / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate