Lin Chien-Huang, Cheng Hui-Wen, Hsu Ming-Jen, Chen Mei-Chieh, Lin Chia-Chin, Chen Bing-Chang
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taiwan.
J Immunol. 2006 Sep 1;177(5):3427-38. doi: 10.4049/jimmunol.177.5.3427.
In this study, we examined the regulation of NF-kappaB activation and IL-8/CXCL8 expression by thrombin in human lung epithelial cells (EC). Thrombin caused a concentration-dependent increase in IL-8/CXCL8 release in a human lung EC line (A549) and primary normal human bronchial EC. In A549 cells, thrombin, SFLLRN-NH2 (a protease-activated receptor 1 (PAR1) agonist peptide), and GYPGQV-NH2 (a PAR4 agonist peptide), but not TFRGAP-NH2 (a PAR3 agonist peptide), induced an increase in IL-8/CXCL8-luciferase (Luc) activity. The thrombin-induced IL-8/CXCL8 release was attenuated by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (a thrombin inhibitor), U73122 (a phosphoinositide-phospholipase C inhibitor), Ro-32-0432 (a protein kinsase C alpha (PKC alpha) inhibitor), an NF-kappaB inhibitor peptide, and Bay 117082 (an IkappaB phosphorylation inhibitor). Thrombin-induced increase in IL-8/CXCL8-Luc activity was inhibited by the dominant-negative mutant of c-Src and the cells transfected with the kappaB site mutation of the IL-8/CXCL8 construct. Thrombin caused time-dependent increases in phosphorylation of c-Src at tyrosine 416 and c-Src activity. Thrombin-elicited c-Src activity was inhibited by Ro-32-0432. Stimulation of cells with thrombin activated IkappaB kinase alphabeta (IKK alphabeta), IkappaB alpha phosphorylation, IkappaB alpha degradation, p50 and p65 translocation from the cytosol to the nucleus, NF-kappaB-specific DNA-protein complex formation, and kappaB-Luc activity. Pretreatment of A549 cells with Ro-32-4032 and the dominant-negative mutant of c-Src DN inhibited thrombin-induced IKK alphabeta activity, kappaB-Luc activity, and NF-kappaB-specific DNA-protein complex formation. Further studies revealed that thrombin induced PKC alpha, c-Src, and IKK alphabeta complex formation. These results show for the first time that thrombin, acting through PAR1 and PAR4, activates the phosphoinositide-phospholipase C/PKC alpha/c-Src/IKK alphabeta signaling pathway to induce NF-kappaB activation, which in turn induces IL-8/CXCL8 expression and release in human lung EC.
在本研究中，我们检测了凝血酶对人肺上皮细胞（EC）中NF-κB激活及IL-8/CXCL8表达的调控作用。凝血酶可使一种人肺EC系（A549）和原代正常人支气管EC中IL-8/CXCL8的释放呈浓度依赖性增加。在A549细胞中，凝血酶、SFLLRN-NH2（一种蛋白酶激活受体1（PAR1）激动肽）和GYPGQV-NH2（一种PAR4激动肽），而非TFRGAP-NH2（一种PAR3激动肽），可诱导IL-8/CXCL8-荧光素酶（Luc）活性增加。凝血酶诱导的IL-8/CXCL8释放可被D-苯丙氨酰-L-脯氨酰-L-精氨酸氯甲基酮（一种凝血酶抑制剂）、U73122（一种磷酸肌醇-磷脂酶C抑制剂）、Ro-32-0432（一种蛋白激酶Cα（PKCα）抑制剂）、一种NF-κB抑制肽以及Bay 117082（一种IκB磷酸化抑制剂）所减弱。凝血酶诱导的IL-8/CXCL8-Luc活性增加可被c-Src的显性负性突变体以及转染了IL-8/CXCL8构建体κB位点突变的细胞所抑制。凝血酶可使c-Src在酪氨酸416处的磷酸化及c-Src活性呈时间依赖性增加。凝血酶引发的c-Src活性可被Ro-32-0432抑制。用凝血酶刺激细胞可激活IκB激酶αβ（IKKαβ）、IκBα磷酸化、IκBα降解、p50和p65从胞质溶胶转位至细胞核、NF-κB特异性DNA-蛋白质复合物形成以及κB-Luc活性。用Ro-32-4032和c-Src的显性负性突变体DN预处理A549细胞可抑制凝血酶诱导的IKKαβ活性、κB-Luc活性以及NF-κB特异性DNA-蛋白质复合物形成。进一步研究表明，凝血酶可诱导PKCα、c-Src和IKKαβ复合物形成。这些结果首次表明，凝血酶通过PAR1和PAR4发挥作用，激活磷酸肌醇-磷脂酶C/PKCα/c-Src/IKKαβ信号通路以诱导NF-κB激活，进而诱导人肺EC中IL-8/CXCL8的表达和释放。
