Thrombin-Induced CCAAT/Enhancer-Binding Protein β Activation and IL-8/CXCL8 Expression via MEKK1, ERK, and p90 Ribosomal S6 Kinase 1 in Lung Epithelial Cells
Thrombin-Induced CCAAT/Enhancer-Binding Protein β Activation and IL-8/CXCL8 Expression via MEKK1, ERK, and p90 Ribosomal S6 Kinase 1 in Lung Epithelial Cells
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DOI：
10.4049/jimmunol.1203323
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发表时间：
2014-01-01
影响因子：
4.4
通讯作者：
Chen, Bing-Chang
中科院分区：
文献类型：
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作者：
Lin, Chien-Huang;Nai, Po-Ling;Chen, Bing-Chang
凝血酶是一种丝氨酸蛋白酶，是血管损伤时产生的凝血因子，在肺部炎症中起重要作用。我们先前发现，c-Src和Rac/PI 3 K/Akt依赖性NF-κ B B通路参与凝血酶诱导的人肺上皮细胞（A549）中IL-8/CXCL 8的表达。在这项研究中，我们研究了MEK激酶（MEKK）1/ERK/p90核糖体S6激酶（RSK）1依赖的C/EBP β信号通路在凝血酶诱导的IL-8/CXCL 8表达中的作用。凝血酶诱导的IL-8/CXCL 8释放和IL-8/CXCL 8荧光素酶活性被C/EBP β的小干扰RNA（siRNA）和用IL-8/CXCL 8构建体的C/EBP β位点突变转染的细胞减弱。此外，凝血酶诱导的κ B-荧光素酶活性也被C/EBP β siRNA抑制。凝血酶诱导的IL-8/CXCL 8释放和IL-8/CXCL 8-荧光素酶的增加也被MEKK 1 siRNA、PD 98059（MEK抑制剂）、U 0126（ERK抑制剂）和RSK 1 siRNA抑制。用凝血酶处理细胞引起Thr（235）处的C/EBP β磷酸化、C/EBP β-荧光素酶活性、C/EBP β向IL-8/CXCL 8启动子的募集以及C/EBP β特异性DNA复合物形成的增加。此外，凝血酶介导的C/EBP β磷酸化和C/EBP β-荧光素酶活性被MEKK 1 siRNA、PD 98059和RSK 1 siRNA抑制。用凝血酶刺激细胞导致RSK 1在Thr（359）/Ser（363）处的磷酸化增加，并且这种作用被MEKK 1 siRNA和PD 98059抑制。凝血酶诱导的ERK激活增加被MEKK 1 siRNA抑制。这些结果表明，凝血酶激活MEKK 1/ERK/RSK 1信号通路，进而启动C/EBP β激活、C/EBP β向IL-8/CXCL 8启动子的募集和C/EBP β特异性DNA复合物的形成，并最终诱导肺上皮细胞中IL-8/CXCL 8的表达和释放。
Thrombin, a serine protease, is a well-known coagulation factor generated during vascular injury and plays an important role in lung inflammation. We previously showed that the c-Src- and Rac/PI3K/Akt-dependent NF-kappa B pathways are involved in thrombin-induced IL-8/CXCL8 expression in human lung epithelial cells (A549). In this study, we investigated the role of the MEK kinase (MEKK)1/ERK/p90 ribosomal S6 kinase (RSK)1-dependent C/EBP beta signaling pathway in thrombin-induced IL-8/CXCL8 expression. Thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity were attenuated by small interfering RNA (siRNA) of C/EBP beta and by cells transfected with the C/EBP beta site mutation of the IL-8/CXCL8 construct. Moreover, thrombin-induced kappa B-luciferase activity was also inhibited by C/EBP beta siRNA. The thrombin-induced increases in IL-8/CXCL8 release and IL-8/CXCL8-luciferase were also inhibited by MEKK1 siRNA, PD98059 (an MEK inhibitor), U0126 (an ERK inhibitor), and RSK1 siRNA. Treatment of cells with thrombin caused an increase in C/EBP beta phosphorylation at Thr(235), C/EBP beta-luciferase activity, recruitment of C/EBP beta to the IL-8/CXCL8 promoter, and C/EBP beta-specific DNA complex formation. Furthermore, thrombin-mediated C/EBP beta phosphorylation and C/EBP beta-luciferase activity were inhibited by MEKK1 siRNA, PD98059, and RSK1 siRNA. Stimulation of cells with thrombin resulted in an increase in RSK1 phosphorylation at Thr(359)/Ser(363), and this effect was inhibited by MEKK1 siRNA and PD98059. The thrombin-induced increase in ERK activation was inhibited by MEKK1 siRNA. These results imply that thrombin activates the MEKK1/ERK/RSK1 signaling pathway, which in turn initiates C/EBP beta activation, recruitment of C/EBP beta to the IL-8/CXCL8 promoter, and C/EBP beta-specific DNA complex formation, and ultimately induces IL-8/CXCL8 expression and release in lung epithelial cells.