title: 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.
abstract: 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-κB 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/EBPβ 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/EBPβ and by cells transfected with the C/EBPβ site mutation of the IL-8/CXCL8 construct. Moreover, thrombin-induced κB-luciferase activity was also inhibited by C/EBPβ 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/EBPβ phosphorylation at Thr(235), C/EBPβ-luciferase activity, recruitment of C/EBPβ to the IL-8/CXCL8 promoter, and C/EBPβ-specific DNA complex formation. Furthermore, thrombin-mediated C/EBPβ phosphorylation and C/EBPβ-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/EBPβ activation, recruitment of C/EBPβ to the IL-8/CXCL8 promoter, and C/EBPβ-specific DNA complex formation, and ultimately induces IL-8/CXCL8 expression and release in lung epithelial cells.
authors.affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan;
authors.forename: Chien-Huang
authors.initials: CH
authors.lastname: Lin
authors.forename: Po-Ling
authors.initials: PL
authors.lastname: Nai
authors.forename: Mauo-Ying
authors.initials: MY
authors.lastname: Bien
authors.forename: Chung-Chi
authors.initials: CC
authors.lastname: Yu
authors.forename: Bing-Chang
authors.initials: BC
authors.lastname: Chen
mesh_terms: D056809:Alveolar Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism; D048728:MAP Kinase Kinase Kinase 1 / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D016209:Interleukin-8; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D048049:Extracellular Signal-Regulated MAP Kinases; D048728:MAP Kinase Kinase Kinase 1; D013917:Thrombin