Ca - and Protein Kinase C-dependent Signaling Pathway for Nuclear Factor- B Activation, Inducible Nitric-oxide Synthase Expression, and Tumor Necrosis Factor- Production in Lipopolysaccharide-stimulated Rat Peritoneal Macrophages*

Lipopolysaccharide (LPS)-activated macrophages are pivotal in innate immunity. With LPS treatment, extracellular signals are transduced into macrophages via Toll-like receptor 4 and induce inflammatory mediator production by activating signaling pathways, including the nuclear factorB (NFB) pathway and the mitogen-activated protein kinase (MAPK) pathway. However, the mechanisms by which the intracellular free Ca2 concentration ([Ca2 ]i) increases and protein kinase C (PKC) is activated remain unclear. Therefore, we investigated the signaling pathway for Ca2 and PKC-dependent NFB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor(TNF) production in LPS-stimulated rat peritoneal macrophages. The results demonstrated that the LPS-induced transient [Ca2 ]i increase is due to Ca2 release and influx. Extracellular and intracellular Ca2 chelators inhibited phosphorylation of PKC and PKC . A PKC -specific and a general PKC inhibitor blunted phosphorylation of serine inmitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK) 1.Moreover, aMEKKinhibitor reducedactivationof inhibitory B kinase and NFB. Upstream of the [Ca2 ]i increase, a proteintyrosine kinase inhibitor reduced phosphorylation of phospholipase C (PLC) . Furthermore, a PLC inhibitor eliminated the transient [Ca2 ]i increase and decreased the amount of activated PKC. Therefore, these results revealed the following roles of Ca2 and PKC in the signaling pathway for NFB activation in LPSstimulated macrophages. After LPS treatment, protein-tyrosine kinasemediates PLC 1/2 phosphorylation, which is followed by a [Ca2 ]i increase. Several PKCs are activated, and PKC regulates phosphorylation of serine inMEKK1. Moreover, MEKKs regulate inhibitory B kinase activation. Sequentially, NFB is activated, and inducible nitric-oxide synthase and tumor necrosis factorproduction is promoted.