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 Thr235, 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 Thr359/Ser363, 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.