Kinetics of mitogen-activated protein kinase family in lipopolysaccharide-stimulated mouse Kupffer cells and their role in cytokine production.
This study was designed to systemically investigate the kinetics of extracellular signal-regulated kinase (ERK) 1/2, p54 c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) in lipopolysaccharide (LPS)-stimulated Kupffer cells (KC) simultaneously at the levels of protein expression, phosphorylation, and kinase activity, respectively, and their role in mediating pro- and anti-inflammatory cytokines. The protein expression, phosphorylation, and activities of these MAPKs in LPS-stimulated primary mouse KCs were determined with SDS-PAGE and western blotting using nonphosphorylated or phosphospecific antibodies or their corresponding substrates. The levels of TNF-alpha and IL-10 in culture supernatants were measured with ELISA kits. The results revealed that LPS stimulation, although not up- or downregulating the protein expression of ERK1/2, p54JNK, and p38 MAPKs in KCs, could induce rapid and significant activation of these kinases, with parallel profiles of changes in both phosphorylation and kinase activities. Although ERK1/2, p54JNK, and p38 kinases in LPS-stimulated KCs have similar kinetics of activation, the activation of ERK1/2 and p38 kinases was the most prominent. Inhibition of p38 MAPK with SB203580 inhibited the production of TNF-alpha and IL-10 by LPS-stimulated KCs, whereas blockade of ERK1/2 with PD98059 could reduce TNF-alpha production, but did not affect IL-10 production. Furthermore, PD98059 and SB203580 had an additive effect on TNF-alpha production, but PD98059 did not augment the SB203580-induced inhibition of IL-10 production. These data indicate that LPS stimulation, although not inducing any change in protein expression, results in rapid activation of ERK1/2, p54JNK, and p38 kinases in KCs, and that they may have different importance in the regulation of pro- and anti-inflammatory responses by LPS-stimulated KCs.
Jiang
Jian-Xin
JX
Research Institute of Surgery, Daping Hospital, The Third Military Medical University, Chongqing, China.
Zhang
Yu
Y
Ji
San-He
SH
Zhu
Peifang
P
Wang
Zheng-Guo
ZG
D000818:Animals; D016207:Cytokines / Q000096:biosynthesis*; D005260:Female; D005419:Flavonoids; D007093:Imidazoles; D007249:Inflammation / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism; D007700:Kinetics; D007728:Kupffer Cells / Q000187:drug effects / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000633:toxicity; D008297:Male; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D011725:Pyridines; D018805:Sepsis / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016207:Cytokines; D005419:Flavonoids; D007093:Imidazoles; D008070:Lipopolysaccharides; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one