Ca2+- and protein kinase C-dependent signaling pathway for nuclear factor-kappaB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-alpha 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-kappaB (NF-kappaB) 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-kappaB 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 PKCalpha and PKCbeta. A PKCbeta-specific and a general PKC inhibitor blunted phosphorylation of serine in mitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK) 1. Moreover, a MEKK inhibitor reduced activation of inhibitorykappaB kinase and NF-kappaB. Upstream of the [Ca2+]i increase, a protein-tyrosine kinase inhibitor reduced phosphorylation of phospholipase C (PLC) gamma. 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 NF-kappaB activation in LPS-stimulated macrophages. After LPS treatment, protein-tyrosine kinase mediates PLCgamma1/2 phosphorylation, which is followed by a [Ca2+]i increase. Several PKCs are activated, and PKCbeta regulates phosphorylation of serine in MEKK1. Moreover, MEKKs regulate inhibitory kappaB kinase activation. Sequentially, NF-kappaB is activated, and inducible nitric-oxide synthase and tumor necrosis factor-alpha production is promoted.
Zhou
Xueyuan
X
Department of Biophysics in the School of Physics, Key Laboratory of Bioactive Materials of Education Ministry, Nankai University, Tianjin 300071, Peoples Republic of China.
Yang
Wenxiu
W
Li
Junying
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D016328:NF-kappa B / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D010537:Peritoneum / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D052247:Nitric Oxide Synthase Type II; D011493:Protein Kinase C; D002118:Calcium