PDK1/RSK1-dependent C/EBPβ activation is involved in thrombin-induced expression of orosomucoid 1-like 3 (ORMDL3) and IL-8/CXCL8 in human lung epithelial cells

Abstract

Asthma is a chronic inflammatory disease of the airways characterized by airway inflammation and structural modeling. Thrombin is known a coagulation factor and shows a critical role in lung inflammation. Previous study demonstrated that overexpressed orosomucoid 1-like 3 (ORMDL3) was strongly linked with asthma and IL-8/CXCL8 release. In this study, we investigated that the role of phosphoinositide-dependent kinase 1 (PDK1), ribosomal S6 kinase 1 (RSK1), and CCAAT/enhancer-binding protein β (C/EBPβ) in thrombin-induced expression of ORMDL3 and IL-8/CXCL8 in human lung epithelial cells (A549). We found that treatment of cells with thrombin caused ORMDL3 mRNA and protein expression. Thrombin-induced IL-8/CXCL8-luciferase activity or IL-8/CXCL8 release was inhibited by ORMDL3 siRNA, BX-912 (a PDK1 inhibitor), and PDK1 siRNA. Moreover, thrombin-stimulated ORMDL3 expression was attenuated by BX-912, PDK1 siRNA, RSK1 siRNA, and C/EBPβ siRNA. Thrombin produced increases in PDK1 phosphorylation at Ser241 in a time-dependent manner. Transfection of cells with PDK1 siRNA or RSK1 siRNA markedly reduced PDK1 or RSK1 protein expression, respectively. Thrombin induced increase in RSK1 phosphorylation at Ser221 in a time-dependent manner, which was inhibited by PDK1 siRNA. Finally, stimulation of cells with thrombin caused an increase in C/EBPβ phosphorylation at Ser105, which was reduced by PDK1 siRNA and RSK1 siRNA. Taken together, these results imply that the PDK1/RSK1/C/EBPβ signaling pathway play a critical role in thrombin-induced ORMDL3 expression and IL-8/CXCL8 expression and release in lung epithelial cells.