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, -ßI, or -
But Not -
Inhibit Lipopolysaccharide-Induced Nitric Oxide Synthase Expression in RAW 264.7 Macrophages: Involvement of a Nuclear Factor 
B-Dependent Mechanism1
Institutes of
*
Pharmacology and
Medical Technology, College of Medicine, National Taiwan University, Taipei, Taiwan
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-
, -ßI,
-
, -
, and -
in RAW cells; down-regulation of PKC-, -ßI,
and -, but not -, was seen after long-term TPA treatment,
indicating the possible involvement of one or all of PKC-, -ßI,
and -, but not -, in LPS-mediated effects. Treatment with
antisense oligonucleotides for these isoforms further demonstrated the
involvement of PKC-, -ßI, and , but not -, in LPS responses.
Stimulation of cells with LPS for 1 h caused activation of NF-
B
in the nuclei by detection of NF-B-specific DNA-protein binding;
this was inhibited by genestein, U73122, D609, calphostin C, or
antisense oligonucleotides for PKC-, -ßI, and -, but not -.
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-B DNA-protein binding, then initiated the expression of iNOS
and NO release. PKC isoforms , ßI, and were shown to be
involved in the regulation of these LPS-induced
events.
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