Different pathways leading to activation of extracellular signal-regulated kinase and p38 MAP kinase by formyl-methionyl-leucyl-phenylalanine or platelet activating factor in human neutrophils
Different pathways leading to activation of extracellular signal-regulated kinase and p38 MAP kinase by formyl-methionyl-leucyl-phenylalanine or platelet activating factor in human neutrophils
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DOI：
10.1007/s11373-005-1704-1
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发表时间：
2005-01-01
影响因子：
11
通讯作者：
Hsu, CM
中科院分区：
文献类型：
--
作者：
Chen, LW;Lin, MW;Hsu, CM
研究了人中性粒细胞细胞外信号调节激酶（ERK）和p38丝裂原活化蛋白激酶（MAPK）被n-甲酰基- met -亮氨酸（fMLP）或血小板活化因子（PAF）激活的信号通路。先前，我们发现PAF和fMLP刺激细胞内Ca2+ ([Ca2+](i))的变化分别是由于Ca2+内流和内部Ca2+释放。为了进一步确定MAPK激活的机制及其与Ca2+内流的关系，我们通过静脉穿刺取健康志愿者的血液。分离人多形核细胞（PMNs），用蛋白激酶C （PKC）抑制剂Calphostin C、PKC- γ异构体抑制剂GF109203X、磷脂酰肌醇3-激酶（PI3K）抑制剂wortmannin和LY294002、磷脂酶C （PLC）抑制剂U73122、磷脂酶A(2) （PLA(2)）抑制剂马铃酸、储存操作钙通道抑制剂SKF96365或细胞外钙螯合剂EGTA孵育，然后用fMLP或PAF处理。免疫印迹法检测ERK p38磷酸化水平。我们的数据表明fMLP和PAF介导的中性粒细胞MAPK信号通路是不同的。paf诱导的ERK磷酸化是由PI3K、PKC、PLA2、PLC和细胞外钙介导的，而fmlp诱导的ERK磷酸化不涉及PKC- γ亚型和细胞外钙。paf诱导的p38磷酸化涉及PLA2，而fmlp诱导的p38激活依赖于PLC。
The signaling pathways leading to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation by N-formyl-Met-Leu-Phe (fMLP) or platelet activating factor (PAF) in human neutrophils were examined. Previously, we found that changes of intracellular Ca2+ ([Ca2+](i)) stimulated by PAF and fMLP were due to Ca2+ influx and internal Ca2+ release, respectively. To further determine the mechanism of MAPK activation and its relation with Ca2+ influx, blood from healthy human volunteers was taken by venous puncture. Human polymorphonuclear cells (PMNs) were isolated and incubated with protein kinase C (PKC) inhibitor Calphostin C, PKC-gamma isoform inhibitor GF109203X, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, phospholipase C (PLC) inhibitor U73122, phospholipase A(2) (PLA(2)) inhibitor Aristolochic acid, store-operated calcium (SOC) channel inhibitor SKF96365, or extracellular calcium chelator EGTA followed by fMLP or PAF treatment. Phosphorylation of ERK p38 was determined by immunoblotting analysis. Our data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK phosphorylation is mediated by PI3K, PKC, PLA2, PLC, and extracellular calcium, whereas fMLP-induced ERK phosphorylation does not involve the PKC-gamma isoform and extracellular calcium. PAF-induced p38 phosphorylation involves PLA2, whereas fMLP-induced p38 activation is PLC dependent.