Switch activation of PI-PLC downstream signals in activated macrophages with wortmannin.
Phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) has been known to serve as a substrate for phosphatidylinositol 3-kinase (PI(3)K) and phosphoinositide-specific phospholipase C (PI-PLC), which can produce PtdIns(3,4,5)P(3) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG), respectively. In this study, we elucidated the role of PI-PLC during the LPS-activated mouse macrophages RAW264.7 treated with PI(3)K inhibitor wortmannin. First, wortmannin treatment enhanced Ins(1,4,5)P(3) production and iNOS expression in LPS-activated macrophages. Inhibition of PI(3)K by p85 siRNA also showed an enhancement of iNOS expression. On the other hand, overexpression of PI(3)K by ras-p110 expression plasmid significantly decreased iNOS expression in LPS-activated macrophages. In addition, overexpression of wild-type or dominant-negative Akt expression plasmid did not affect the iNOS expression in LPS-activated macrophages. Second, treatment of PI-PLC inhibitor U73122 reversed the enhancement of iNOS expression, the increase of phosphorylation level of ERK, JNK and p38, and the increase of AP-1-dependent gene expression in wortmannin-treated and LPS-activated macrophages. However, NF-kappaB activity determined by EMSA assay and reporter plasmid assay did not change during LPS-activated macrophages with or without wortmannin. We propose that the inhibition of PI(3)K by wortmannin in mouse macrophages enhances the PI-PLC downstream signals, and subsequently increases the LPS induction of iNOS expression independently of Akt pathway.
Liu
Der-Zen
DZ
Graduate Institutes of Biomedical Materials, Taipei Medical University, Taipei, Taiwan.
Liang
Hong-Jen
HJ
Chen
Chien-Ho
CH
Lin
Shyr-Yi
SY
Zhong
Wen-Bin
WB
Ho
Feng-Ming
FM
Hou
Wen-Chi
WC
Lo
Jui-Lien
JL
Ho
Yuan-Soon
YS
Lin
Pei-Jung
PJ
Hung
Ling-Fang
LF
Liang
Yu-Chih
YC
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D004075:Diglycerides / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D054801:Phosphoinositide Phospholipase C; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D004075:Diglycerides; D008070:Lipopolysaccharides; D016328:NF-kappa B; D019269:Phosphatidylinositol 4,5-Diphosphate; D010726:Phosphodiesterase Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D018808:Transcription Factor AP-1; D015544:Inositol 1,4,5-Trisphosphate; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D000077191:Wortmannin