Ca2+- and protein kinase C-dependent signaling pathway for nuclear factor-κB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-α 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 factor-kappa B (NF-kappa B) 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 NF-kappa B activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-alpha (TNF-alpha) 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 alpha and PKC beta. A PKC beta-specific and a general PKC inhibitor blunted phosphorylation of serine in mitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK) 1. Moreover, aMEKK inhibitor reduced activation of inhibitory kappa B kinase and NF-kappa B. Upstream of the [Ca2+](i) increase, a proteintyrosine kinase inhibitor reduced phosphorylation of phospholipase C (PLC) gamma. Furthermore, a PLC inhibitor eliminated the Ktransient [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 NF-kappa B activation in LPS-stimulated macrophages. After LPS treatment, protein-tyrosine kinase mediates PLC gamma 1/2 phosphorylation, which is followed by a [Ca2+](i) increase. Several PKCs are activated, and PKC beta regulates phosphorylation of serine in MEKK1. Moreover, MEKKs regulate inhibitory kappa B kinase activation. Sequentially, NF-kappa B is activated, and inducible nitric-oxide synthase and tumor necrosis factor-alpha production is promoted.