Antisense oligonucleotides targeting protein kinase C-alpha, -beta I, or -delta but not -eta inhibit lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages: involvement of a nuclear factor kappa B-dependent mechanism.
The signaling pathway for protein kinase C (PKC) activation and the role of PKC isoforms in LPS-induced nitric oxide (NO) release were studied in RAW 264.7 macrophages. The tyrosine kinase inhibitor genestein attenuated LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression, as did the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122 and the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609. LPS stimulated phosphatidylinositol (PI) hydrolysis and PKC activity in RAW cells; both were inhibited by genestein. The PKC inhibitors (staurosporine, calphostin C, Ro 31-8220, or Go 6976) or long-term 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment also resulted in inhibition of LPS-induced NO release and iNOS expression. Western blot analysis showed expression of PKC-alpha, -betaI, -delta, -eta, and -zeta in RAW cells; down-regulation of PKC-alpha, -betaI, and -delta, but not -eta, was seen after long-term TPA treatment, indicating the possible involvement of one or all of PKC-alpha, -betaI, and -delta, but not -eta, in LPS-mediated effects. Treatment with antisense oligonucleotides for these isoforms further demonstrated the involvement of PKC-alpha, -betaI, and delta, but not -eta, in LPS responses. Stimulation of cells with LPS for 1 h caused activation of NF-kappaB in the nuclei by detection of NF-kappaB-specific DNA-protein binding; this was inhibited by genestein, U73122, D609, calphostin C, or antisense oligonucleotides for PKC-alpha, -betaI, and -delta, but not -eta. These data suggest that LPS activates PI-PLC and PC-PLC via an upstream tyrosine kinase to induce PKC activation, resulting in the stimulation of NF-kappaB DNA-protein binding, then initiated the expression of iNOS and NO release. PKC isoforms alpha, betaI, and delta were shown to be involved in the regulation of these LPS-induced events.
Chen
C C
CC
Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei. ccchen@ha.mc.ntu.edu.tw
Wang
J K
JK
Lin
S B
SB
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D016328:NF-kappa B / Q000096:biosynthesis / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D015398:Signal Transduction / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008070:Lipopolysaccharides; D016328:NF-kappa B; D016376:Oligonucleotides, Antisense; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C497259:Prkcd protein, mouse; C081975:protein kinase C eta; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate