Phorbol ester synergistically increases interferon regulatory factor-1 and inducible nitric oxide synthase induction in interferon-gamma-treated RAW 264.7 cells.
The roles of PKC in iNOS induction by IFN-gamma have been shown in some cell types. The effect of a PKC activator, phorbol ester, in iNOS induction is thought to be due to multiple mechanisms, and it is necessary to examine the involvement of phorbol ester on IFN-gamma-induced iNOS in detail. In the present study, we investigated the mechanisms of phorbol ester on IFN-gamma-induced iNOS in RAW 264.7 cells. PMA synergistically increased iNOS activity, protein and mRNA levels in IFN-gamma-treated RAW 264.7 cells. PMA together with IFN-gamma increased iNOS mRNA without affecting the iNOS mRNA degradation, suggesting that the synergistic effect of PMA on IFN-gamma-induced iNOS mRNA production may depend on the elevation of the transcription rate rather than a prolongation of mRNA stability. The DNA binding proteins that are involved in the regulation of iNOS expression are mainly NF-kappa B and IRF-1. IRF-1 transcriptionally regulates many IFN-inducible genes such as iNOS whose promoter contains an IRF-1 binding site. PMA might modulate iNOS induction as a cosignal with IFN-gamma in RAW 264.7 cells because the synergistic effect of PMA was mediated through IRF-1, rather than NF-kappa B. Ro 31-8220, a PKC inhibitor, decreased iNOS activity, protein, mRNA levels and IRF-1 activity, indicating that the effect of PMA on iNOS induction might occur via the PKC pathway. It is evidence that PKC plays an important role in IRF-1 activation and that phorbol ester has a synergistic effect on iNOS induction through IRF-1 activation in IFN-gamma-treated RAW 264.7 cells. The synergistic effect of PMA on IFN-gamma-induced IRF-1 binding activity was observed in macrophage cell line J774 cells as well as RAW 264.7 cells, but not in thioglycollate-elicited peritoneal macrophages.
Momose
I
I
Department of Psychiatry, Shimane Medical University, Izumo, Japan.
Terashima
M
M
Nakashima
Y
Y
Sakamoto
M
M
Ishino
H
H
Nabika
T
T
Hosokawa
Y
Y
Tanigawa
Y
Y
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000494:pharmacology*; D051379:Mice; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D010703:Phorbol Esters / Q000494:pharmacology*; D010750:Phosphoproteins / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D010703:Phorbol Esters; D010750:Phosphoproteins; D012333:RNA, Messenger; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D011493:Protein Kinase C; C064758:Ro 31-8220